Genel Bilgi:

D Vitamininin başlıca etkileri arasında intestinal kalsiyum emilimini arttırmak, PTH gen transkripsiyonunu inhibe etmek, kemik matriks proteinlerinin ekspresyonunu düzenlemek ve osteoklast farklılaşmasını hızlandırmak bulunmaktadır. D vitamini yetersizliğine bağlı klinik problemler olan rikets ve osteomalazi günümüzde önemini korumakla birlikte D vitamini, son yıllarda esas olarak “subklinik D vitamin eksikliği” tanımı çerçevesinde ve iskelet sistemi dışındaki etkileri nedeniyle güncelleşmiştir.

Bunun yanında D vitamin düşüklüğü özellikle sekonder hiperparatiroidizm gelişen bireylerde olmak üzere osteporoz gelişimi için önemli bir risk faktörüdür; ayrıca kas güçsüzlüğüne(ve buna bağlı düşmelere) neden olarak kırık riskini arttırmaktadır. D vitamin düşüklüğü kanser, diyabet ve diğer otoimmün hastalıklar için risk faktörüdür. Bu nedenle erişkin sağlığı bakımdan yeterli D vitamini düzeyine sahip olmak giderek önem
kazanmaktadır.

İnsan vücudunda D vitamini durumu 25-hidroksivitamin D 10–30 (25-OH D) düzeyi ile
değerlendirilmektedir. Serum 25-OH D düzeyi güneş ışığına maruziyet, yaşanılan bölgenin deniz seviyesinden yüksekliği, deri pigmentasyon yoğunluğu, yaş ve beslenmeyle alınan D vitamini miktarına göre değişmektedir. Bu nedenlere bağlı değişiklikler göstermekle birlikte genel olarak; erişkinlerde paratiroid hormon (PTH) yükselmesine neden olmayacak 25-OH D düzeyi olan 30 ng/ml eşik değer olarak alınmakta ve 30 ng/ml altındaki değerler yetersiz/düşük, 10 ng/ml altı ise eksiklik olarak normal kabul edilmektedir.

Perinatal D vitamin eksikliği ve etkileri:

Son 20 yılda dikkatler anne ve bebeğin biyolojik birliği temelinde D vitamini eksikliğinin anne ve bebeğin ortak bir sorunu olduğuna yoğunlaşmış ve bu çerçevede perinatal D vitamini eksikliği tanımlaması önem kazanmıştır. Maternal D vitamini eksikliğinin yenidoğan ve bebeklik dönemindeki D vitamini eksikliği ve “infantil rikets” için en önemli risk faktörü olmasının yanı sıra, D vitamininin özellikle kemik dışı etkileri göz önüne alındığında gebelik döneminin kritik bir dönem olabileceği, gebelikteki D vitamin eksikliğinin fetus üzerindeki etkilerinin yaşam boyu sürebileceği üzerinde durulmaktadır.

Fetus kalsiyum ihtiyacını anneden karşılar; annenin gebelikte ve laktasyon döneminde normak kalsiyum dengesi için D vitamini düzeyinin yeterli olması gereklidir. Doğumla birlikte anneden sağlanan kalsiyum kesilir ve ilk günlerde bebekler PTH ve aktif D vitamin ile kendi kalsiyum dengelerini korumaya çalışır. 25-OHD plasentayı geçebilir ve yenidoğanlar doğumda annelerin sağladığı D vitamin desteğine bağımlıdır. Ülkemizde yapılan bir çalışmada yenidoğanlarda düşük 25-OHD düzeyi için en önemli risk faktörünün anne 25- OHD düzeyinin <10 ng/ml olması ( (OR = 15.2, p = 0.02) gösterilmiştir. Bu nedenle D vitamini eksikliği olan annelerden, doğan bebeklerde eğer dışardan destek sağlanmazsa serum 25-OHD düzeyleri hızla düşer ve bu da yenidoğan döneminde hipokalsemi ve/veya konjenital riketse neden olur. Annedeki D vitamin eksikliğinin şiddetli olduğu durumlarda bebeklere verilen 400 IU D vitamin ile yenidoğan dönemindeki hipokalsemileri önlemek mümkün olmayabilir. Ayrıca bebeklere ilk hafta içinde D vitamin başlanması mümkün olmayabilir. Dolayısı ile erken yenidoğan döneminde D vitamin eksikliğinin önlenmesi ancak annelerin D vitamin düzeyinin yeterli olması ile sağlanabilir. Benzer şekilde anne sütündeki 25-OHD düzeyi de annenin D vitamini durumundan etkilenmekte ve bebeklerdeki D vitamin eksikliği için risk faktörü olarak kabul edilmektedir.

Maternal D vitamin eksikliği ve etkileri :

Son yıllarda yapılan çalışmalarda; maternal D vitamin eksikliğinin neonatal hipokalsemi ve infantil rikets yanında eklampsi/preeklampsi, fizyolojik kraniotabes sıklığında artma, düşük doğum ağırlığı/prematür doğum, dental/enamel hipoplazi, konjenital katarakt, çocuklarda Tip 1 diyabet, multiple skleroz, bipolar bozukluk, depresyon, astım, mental retardasyon sıklığında artma gibi sorunlarla ilişkili olabileceği üzerinde durulmaktadır.

Ayrıca D vitamininin fetal beyin ve immün sistem gelişimini etkilediği ileri sürülmektedir. Gebelikte D vitamini yetmezliğinin yenidoğan üzerindeki etkileri kalıcı olabilir ve daha sonradan verilecek D vitamin desteği ile tam olarak düzeltilemeyebilir. Bu durum özellikle beyin ve immün sistem gelişiminde önemlidir.

Yapılan araştırmalarda; D vitamin seviyesi düşük olan annelerde sezaryen oranı yüksek bulunmuştur. Preeklampsi riski 5 kat, gestasyonel diyabet riski 3 kat, bakteriyel vajinozis riski 2 kat artmış bulunmuştur.

Maternal D vitamini eksikliğinin anne ve bebekler üzerindeki etkisi aşağıda yer alan Tablo’da özetlenmiştir.

D Vitamini eksikliğini önlemek için yapılması gerekenler:

Ülkemizde gebelik ve süt verme döneminde annelere D vitamin desteği sağlanması hem anne sağlığı bakımından hem de bebeklerde D vitamini eksikliğinin önlenmesi bakımından gereklidir. Bakanlığımız da gebelerde D vitamini eksikliğinin yaygın bir sorun olmasını dikkate alarak ücretsiz D vitamini dağıtılmasına dayanan bir program başlatmıştır.

Programın aşağıdaki şekilde uygulanması gerekmektedir.

1. Gebelere D vitamini başlanırken; ülkemizde değişik bölgelerde yapılan çok geniş ölçekli olmayan çalışmaların sonuçları, D Vitamini eksikliğinin sıklığı ve şiddeti göz önüne alındığında gebelerde kan düzeyine bakılmaksızın başlanır.
2. Gebeye uygulanacak D vitamini desteği, gebeliğin 12. haftasından itibaren başlanmalı, gebelik süresince anneye destek sağlanmalı, doğum sonrası 6 ay sürdürülmelidir.
3. Doğum öncesi dönemde gebelere ve doğumdan sonraki dönemde annelere uygulanacak D vitamini dozu günlük tek doz olarak alınmak üzere 1200 IU (9 damla) olmalıdır.
4. D Vitamini desteği başlanan anne; hiperkalsemi bulguları hakkında (iştahsızlık, bulantı, kabızlık, poliüri, polidipsi vb) bilgilendirilmeli, izlenmeli ve değerlendirilmelidir. Gerekli durumlarda serum Ca, serum 25-OHD ve spot idrarda kalsiyum düzeyi bakılması için sevk edilmelidir.
5. D vitamini damlası içeren preparat; program kapsamında ödeme gücü olmayanlar için ücretsiz olarak temin edilecek sosyal güvencesi olanlar için ise reçete edilecektir.
6. Gebeler, ek olarak önerilen D vitamin damlasının yanı sıra önerilen multivitamin içerikli ilaçları kullanmaya devam edebileceklerdir.

ABSTRACT

Maternal hypothyroidism and hypothyroxenemia due to iodine deficiency have been shown to affect development of the newborn negatively. Maternal iodine supplementation may therefore improve cognitive performance of the offspring, even in areas of mild-to-moderate iodine deficiency (ID). Several
iodine supplementation studies have been performed in mildly ID pregnant women in Europe.These
studies have shown that iodine supplementation increases maternal urinary iodine (UI) excretion and
reduces thyroid volume, as well as prevents increases in infant thyroid volume and thyroglobuline.

However, randomized controlled studies with long-term outcomes are lacking. Therefore, two trials were
started in 2008 in areas of low iodine status; one in Bangalore, India (n = 325), and another in Bangkok,
Thailand (n = 514). Pregnant women were recruited <14 weeks gestational age and randomized to either receive a daily dose of 200 g I (as KI) or an identical placebo throughout pregnancy.

Both trials are ongoing, and women are followed up during pregnancy and at delivery.UI, thyroid hormones, and thyroid size are measured. Birth outcomes are recorded, such as gestational age at delivery, height, weight, and APGAR scores, and cord blood and heel stick blood (<72 h) is collected from the child. Child development is assessed at 6 weeks of age using the Neonatal Behavioral Assessment Scale (NBAS), and at 12 and 24 months of age using the Bayley Scales of Infant Development. The outcomes of these trials will contribute importantly to the evidence base for iodine supplementation of pregnant women living in areas of mild iodine deficiency.

Iodine deficiency and its consequences for brain development

Iodine has since long been recognized an important element for fetal growth and development. Severe iodine deficiency during pregnancy is the cause of congenital hypothyroidism resulting in cretinism, an irreversible form of retarded growth and mental retardation.

Cretinism used to be common in mountainous areas, such as the Alps, the Andes and the Himalaya where glaciers and erosion have depleted soils from iodine. Cretinism, however, is confined only to a small group of individuals in a deficient population.

A much larger proportion of a population living in an iodine deficient area is exposed to milder consequences, generally referred to as the iodine deficiency disorders (IDD). This comprises hypothyroidism and goiter, but also impaired cognition.

It has been estimated that iodine deficiency ranks third on the list by Bleichrodt and Born states that moderate to severe iodine deficiency leads to a reduction of 12–13.5 IQ points in children. A major limitation is that primarily cross-sectional studies were included in this meta-analysis, which therefore may have suffered from confounding. Moreover, many of the studies included were not peer reviewed, and no rigorous exclusion criteria were used. Many more studies with similar outcomes have been performed since, also in areas with less severe iodine deficiency.

Yet, confounding of the study results has mostly not been ruled out. It is also not always clear from these studies whether the reduction in IQ should be attributed to iodine deficiency in utero, or to iodine deficiency in later life. Intervention studies in school children do suggest, on the whole, that cognitive impairment related to mild-to-moderate iodine deficiency is, at least partly, reversible.

Should pregnant women in areas with mild deficiency be advised to take iodine supplements?

Since brain development starts very early in fetal life, it may well be that iodine has its most significant effect if consumed by expectant mothers in sufficient amounts early in pregnancy. The current WHO guidelines suggestthatiodized salt, provided thatitis universally available (i.e. 90% of households have access to iodized salt), will provide sufficient iodine for women during pregnancy.

Urinary iodine concentrations in school children are recommended as an indicator for iodine status of the total population. However, as we have previously shown in a study comparing UI in pairs of pregnant women and their school-aged children in Thailand, there is reason to believe that children receive sufficient iodine with salt iodization, whereas their mothers do not.

In that case,targeted supplementation of pregnant women would be a good additional strategy, even in areas where iodized salt has already been implemented. Additional studies in this and other settings are required to investigate this further.

Iodine supplementation studies in mildly deficient pregnant subjects mostly stopped before or at the moment of delivery. These studies, all conducted in mildly deficient areas in Europe, have shown that iodine supplementation increases maternal urinary iodine (UI) excretion and reduces thyroid volume, as well as prevents increases in infant thyroid volume and thyroglobuline.

In general, these studies suggest that maternal thyroid metabolism can cope with mild iodine deficiency. Effects of maternal iodine supplementation on infant growth and development, however, have not yet been adequately addressed. Three non-randomized studies conducted in Spain, a country with historic mild iodine deficiency, have been published in which children from supplemented and non-supplemented women were followed and tested over time.

In one of these studies, children 18 months old born to women with low fT4 levels, who were supplemented either from early gestation (4–6 weeks, n = 13), later gestation (12–14 weeks, n = 12) or after delivery (n = 19), were tested for their developmental quotient using the Brunet–Lézine scale. There was a significant trend in developmental quotient over the three groups, with the group longest exposed to iodine supplementation having the highest developmental quotient (101 vs. 92 vs. 87, P < 0.05) .

In another study, children from a group of pregnant women who had received iodine supplements from the first trimester of pregnancy onwards (n = 133) were compared with children from mothers who had not received iodine supplements (n = 61). Children were assessed by the Bayley Scales ofInfant Development at the age of 2 years. Children from supplemented mothers had a 6.1 point higher Psychomotor Development Index score (P < 0.02), but the Mental Development Index score did not differ between the two groups. In a third study, total iodine intake, both from diet and supplements, was assessed in a group of
pregnant women and related to the PDI and MDI of their infants at one year of age. In contrast to the two other studies, it was found that children from mothers with high intake of iodine from supplements (>150 g/d) had a 5.2 lower PDI score.

Apart from the inconsistencies in outcomes between these studies, neither is equipped to provide hard evidence for any beneficial or harmful effects of maternal iodine supplementation on the mental and motor development of their offspring.

It is therefore still a question mark whether iodine supplementation during pregnancy should be recommended in areas of mild-to-moderate iodine deficiency. In order to investigate this, we have set up randomized controlled trials at two study sites (Bangkok, Thailand; and Bangalore, India) with the objective to determine the effects of daily oral iodine supplementation or placebo in pregnant women with mild-to-moderate ID on maternal and infant thyroid function, birth outcome and child development (the MITCH studies). Recruitment began in 2008. Pregnant women, <14 weeks of gestation, aged 18–40 years with singleton pregnancies and not receiving iodine supplements have been included in the study.

Women with overt hypothyroidism (TSH > 6 mIU/L) have been excluded. Eligible women have been randomly assigned to receiving daily iodine supplements (200KI) or identical placebo tablets until delivery. Pregnant women are followed up twice during pregnancy and at delivery. In total, 514 women have been included in Thailand, and 325 in India.

Data collected from mothers include urinary iodine and thyroid hormone concentrations, as well as thyroid volume. Cord blood and heel stick blood (<72 h) is collected from the neonates for assessment of thyroid function. Other data collected from the infants include birth weight and height, Apgar scores, urinary iodine, and thyroid volume.

The Neonatal Behavioral Assessment Scale (NBAS) is performed around 6 weeks of age, and the MDI and PDI are assessed with the Bayley Scales of Infant Development at 12 and 24 months of age. The last data are expected to be collected in November 2013.

In conclusion, up till now it is unknown whether maternal iodine supplementation in areas of mild-to-moderate iodine deficiency should be recommended. Outcomes of the ongoing trials are awaited to prove or disprove the safety and efficacy of iodine supplementation in pregnancy.

DIETARY IODINE INTAKE IS OBLIGATORY FOR THE PRODUCTION OF THYROID HORMONES.

Despite substantial public health advances over the past 3 decades, iodine deficiency currently affects 1.92 billion people globally.1 Dietary iodine requirements are increased during pregnancy due to increased thyroid hormone production, increased renal iodine losses, and fetal iodine requirements.

Dietary requirements remain increased in lactation due to the concentration of iodine in breast milk. Adverse effects of iodine deficiency in pregnancy, when the deficiency leads to severe decreases in maternal thyroxine (T4), include maternal and fetal goiter, cretinism, intellectual impairments, neonatal hypothyroidism, and increased pregnancy loss and infant mortality.

Decreases in maternal T4 associated with even mild iodine deficiency may have adverse effects on the cognitive function of offspring, and iodine deficiency remains the leading cause of preventable intellectual disability worldwide.

Iodine Status in the United States

Urinary iodine values are used most frequently to screen for iodine deficiency in populations. Identifying specific individuals at risk for iodine deficiency is not possible due to diurnal and day-to-day variation in urinary iodine excretion. According to World Health Organization (WHO) and International Council for the Control of Iodine Deficiency Disorders (ICCIDD) guidelines, median urinary iodine values for pregnant women between 150 and 249 g/L (to convert to nmol/L, multiply by 7.880) are consistent with optimal iodine intake.3 US iodine intake decreased by half between the 1970s and 1990s. In the most recent national survey, the median urinary iodine level for pregnant women was only 125 g/L, suggesting that mild iodine deficiency occurs in at least a subset of pregnant US women.

Recommendations for Iodine During Pregnancy and Lactation

The US Institute of Medicine’s recommended dietary allowance for iodine is 220 g per day during pregnancy and 290 g per day during lactation, higher than the 150 g per day recommended for nonpregnant adults.5 Similarly, WHO and ICCIDD guidelines suggest an iodine intake of 200 to 300 g per day for pregnant and lactating women.

Given the decrease in median urinary iodine concentration in the United States and the importance of iodine during pregnancy, the American Thyroid Association (ATA)6 and the Neurobehavioral Teratology Society7 recommend that all US women who are pregnant, lactating, or planning a pregnancy should ingest dietary supplements containing 150 g of potassium iodide per day.

The Endocrine Society has recently advocated that all daily prenatal multivitamins should contain 150 to 200 g.8 The addition of 150 g of potassium iodide does not pose a risk, even for women who are iodine replete, because a total iodine intake of as much as 500 to 1100 g per day is considered safe in pregnancy. Nevertheless, 49% of the different types of prenatal multivitamin brands marketed in the United States contain no iodine9 and it is estimated that only 20% of pregnant women in the United States use iodine-containing supplements.

The Iodine Conundrum

Many pregnant women in the United States do not receive iodine supplementation during pregnancy or lactation despite current recommendations. The adverse effect of severe iodine deficiency has been well described, along with the beneficial effects of iodine supplementation programs on obstetric outcomes and infant health and development.

However, the data regarding iodine supplementation in mildly iodine deficient pregnant women are less robust. Studies suggest that such supplementation is associated with increased maternal urine iodine concentrations, decreased maternal and neonatal thyroid volumes, and decreased neonatal thyroglobulin (a marker of iodine sufficiency).

Two of the 3 prospective studies assessing the effects of iodine supplementation for mildly iodine deficient pregnant women have demonstrated improvements in child cognition but all are limited due to lack of randomization and small sample sizes. The iodine status of pregnant and lactating women in the United States over the next decade is difficult to predict.

Nevertheless, recent trends in the United States suggest that median urinary iodine levels will remain constant at best, or may decline further. Public health efforts to limit salt intake to decrease cardiovascular risk, in conjunction with increasing use of kosher salt and sea salt (neither of which contain iodine), may adversely affect median urinary iodine levels. Furthermore, decreasing use of iodate conditioners in bread products and iodophor cleansers in the dairy
industry may exacerbate the problem.

The Public Health Conundrum Concerning Iodine and Pregnancy

Median urinary iodine levels in pregnant women have experienced a 50% decline. Many pregnant women in the United States, for whom adequate iodine status is essential for the neurocognitive health of the developing fetus, already have iodine levels less than the lower limit of normal as recommended by the WHO and ICCIDD. The effect of mild maternal iodine insufficiency on fetal health has been shown but studies in this area are limited.

However, a randomized placebo-controlled interventional trial in which
some pregnant women do not receive iodine is unethical because increased iodine intake during pregnancy is already recommended by the WHO, ICCIDD, the ATA, the Endocrine Society, and the American Congress of Obstetricians and Gynecologists. What are the appropriate next steps? The status quo can be maintained in which iodine supplementation is recommended, but provided in only 50% of prenatal vitamins.

The alternative is for relevant medical organizations (the ATA, Endocrine Society, American Congress of Obstetricians and Gynecologists, the Council for Responsible Nutrition, and others) to work collaboratively with pharmaceutical and vitamin manufacturers to ensure that all prenatal multivitamins contain 150 g of potassium iodine. In the interim, clinicians should recommend only those prenatal vitamins that contain iodine. The path seems clear. It is time for all prenatal vitamins to contain iodine.

Coenzyme Q10 supplementation during pregnancy reduces the risk of pre-eclampsia

Abstract

Objective: To assess whether supplementation with Coenzyme Q10 (CoQ10) during pregnancy reduces the risk of pre-eclampsia. Methods: Women at increased risk of pre-eclampsia were enrolled in a randomized, double-blind, placebo-controlled trial. Women were assigned to receive 200 mg of CoQ10 or placebo daily from 20 weeks of pregnancy until delivery. The primary outcome was rate of pre-eclampsia. Statistical
analyses were by intention-to-treat. Results: Of the 235 women enrolled in the trial, 118 were randomized to receive CoQ10 and 117 received a placebo. A total of 197 (83.8%) women were followed-up. The overall rate of pre-eclampsia was 20% (n= 47). Thirty women (25.6%) in the placebo group developed pre-eclampsia compared with 17 women (14.4%) in the CoQ10 group, and this reduction was significant (P= 0.035) (relative risk [RR] 0.56; 95% confidence interval [CI], 0.33–0.96). Conclusion: Supplementation with CoQ10 reduces the risk of developing pre-eclampsia in women at risk for the condition.

© 2008 International Federation of Gynecology and Obstetrics.
Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Pre-eclampsia is a common disorder of human pregnancy (about 7% of all pregnancies) in which the normal hemodynamic response to pregnancy is compromised.

It remains a leading cause of maternal morbidity and mortality and is associated with a significant increase in perinatal mortality.

The pathogenesis of pre-eclampsia is still not fully understood, but it is generally accepted that the placenta is implicated in the production of a generalized maternal inflammatory response, which is characterized by activation of maternal vascular endothelial cells and leukocytes.

Moreover, in patients with preeclampsia, there is an increase in the rate of lipid peroxidation, an increase in lipid availability, and a decrease in the levels of antioxidants such as alpha-tocopherol, ascorbate, beta-carotene, and selenium.

Coenzyme Q10 (CoQ10) is an essential component of oxidative phosphorylation at mitochondrial level, and also functions to stabilize cell membranes as well as acting as a potent antioxidant. CoQ10 is involved in pathological conditions such as cancer, cardiovascular diseases, and mitochondrial and muscular diseases.

In 2003, we reported that pregnant women with established preeclampsia had significantly lower plasma levels of CoQ10 compared with healthy pregnant and nonpregnant women. These findings have been confirmed by other cohort studies.

However, these data were collected from women with established pre-eclampsia and, therefore, it was not possible to determine whether the biochemical changes were a cause or a consequence of pre-eclampsia. The aim of the present study was to investigate whether supplementation with CoQ10 during pregnancy reduced the risk of pre-eclampsia in women
at increased risk for the condition.

2. Materials and methods

A randomized, double-blind, placebo-controlled trial was conducted between March 6, 2004 and August 27, 2006.

The trial recruited women with clinical risk factors for pre-eclampsia (primigravidas, 20 years or younger, and residing in Quito which is at 2800 m altitude).

Inclusion criteria were women between 16 and 20 weeks of pregnancy (established by date of last menstrual period and confirmed by ultrasound), not currently taking medication and with no known medical disorders, who attended the prenatal clinic at the Hospital Gineco Obstetrico Isidro Ayora, Quito, Ecuador.

Women who were taking vitamin supplements were not excluded from the trial. Participants were evaluated every 4 weeks until the 36th week, and then every 2 weeks up to delivery. Whenever possible trial visits coincided with routine prenatal appointments. The study was approved by the Bioethics Committee at the Biomedical Center, Central University of Ecuador.

Written informed consent was obtained from all participants. To ensure double-blind allocation the principle investigator, who was not directly involved with the clinical team, used a random number sequence generated by computer software (Epi Info 6.04, Bethesda, MD, USA) to allocate the participants to either the treatment or placebo group.

Neither the trial staff nor the participants knew the treatment allocation. Women randomized to the treatment group received 100-mg softgel capsules containing an enhanced-absorption  CoQ10 formula (Q-absorb; Jarrow Formulas, Los Angeles, CA, USA).

Women in the placebo group received identical-looking softgel capsules produced by the same manufacturer. Both products were packaged in identical plastic bottles identifiable only by the patient all location number. Each patient was provided with a bottle containing a 1-month supply of trial medication.

The women were told to take 2 softgels each day (1 in the morning and 1 in the afternoon). Acceptable compliance was defined as taking at least 80% of the softgels. The women were instructed to leave all remaining softgels in the bottle so that they could be counted later.

At the start of the trial, a 10 mL venous blood sample was collected from each participant to determine baseline plasma levels of CoQ10 and total cholesterol. A 10 mL blood sample was then collected from each trial participant every 4 weeks up to the 36th week and then every 2 weeks until delivery. Samples taken at delivery were obtained when the women were admitted to hospital, before labor started. The samples were immediately transferred into heparinized polypropylene vials and gently mixed by inversion.

Samples were centrifuged at 4 °C for 10 minutes at 1200 ×g; the plasma fraction was transferred into another vial and stored in 500 μL aliquots at –40 °C until assayed as previously described. Samples were measured in duplicate and the mean value was used.

Plasma cholesterol was determined in duplicate samples using a spectrophotometer (Eppendorf, Hamberg, Germany) and a Cholesterol Liquicolor test kit (Human GmbH, Wiesbaden, Germany).

The primary outcome in the study was pre-eclampsia, defined as a blood pressure greater than 140/90 mm Hg on at least 2 occasions more than 6 hours apart and proteinuria greater than 300 mg/dL (2+ or greater by dipstick on 2 occasions 4–24 hours apart) appearing in the second trimester of pregnancy.

We estimated that a sample size of 206 women would have a statistical power of 80% (2-tailed alpha level of 0.05) to detect a reduction in the risk of pre-eclampsia among the women from 20% to 6%. Data are presented by group as number (%) or mean± SD as appropriate.

Primary outcome (occurrence of pre-eclampsia) is reported by intention-to-treat analysis using the Fisher exact test. Plasma levels of CoQ10 in each group were compared using 1-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test. Differences in CoQ10 levels between the treatment and placebo group were compared using unpaired t test. All analyses were performed using GraphPad InStat version 3.01 for Windows (GraphPad Software, La Jolla, CA, USA). Pb0.05 was considered

 

 

significant. The study was registered at: www.clinicaltrials.gov
No. TNC00300937.

3. Results

Of 235 women initially enrolled in the trial, 197 (83.8%) attended at least 2 visits and were followed-up  On the basis of the pill count, 154 women (65.5%) were compliant; the percentage was significantly higher in the CoQ10 supplementation group (85.1% vs 71.9%, P= 0.02). Less than 10% of women were taking multivitamin supplements at enrolment. The last woman delivered on September 3, 2006. The demographic characteristics at enrollment did not differ between women in the CoQ10 group and those in the placebo group. Mild gastrointestinal symptoms were the most common adverse effects, but the difference was not significant between the groups (1.3% CoQ10 vs 1.5% placebo; P= 0.84). Only 2 pregnancies resulted in preterm delivery (before 36 weeks of pregnancy) and both were in the placebo group. The incidence of low birth weight (less than 2500 g) was similar in the placebo group (12.3%) compared with the CoQ10 group (10.0%). There was no perinatal mortality. Mean birth weight was 2981± 387 g in the CoQ10 group compared with 2938± 396 g in the placebo group; and the average duration of pregnancy was 39.1± 1.4 weeks in the CoQ10 group compared with 39.1± 1.5 weeks in the placebo group. Neither birth weight nor pregnancy duration was significantly different between the groups. The overall rate of pre-eclampsia was 20% (n= 47); 17 women in the CoQ10 group and 30 in the placebo group. Supplementation with CoQ10 significantly reduced (P= 0.035) the risk for pre-eclampsia from 25.6% in the placebo group to 14.4% in the CoQ10 group (relative risk [RR] 0.56; 95% confidence interval [CI], 0.33–0.96). Baseline levels of CoQ10 (corrected by plasma cholesterol) were similar in the CoQ10 and placebo groups (Table 1). Plasma levels of CoQ10 increased significantly compared with the baseline value (Pb0.0001) at each assessment stage in the CoQ10 group. However, the increase was not significant compared with the levels

 

 

obtained at 24 weeks. This finding was independent of the development of pre-eclampsia . Plasma concentrations of CoQ10 in thewomen in the placebo group who developed pre-eclampsia showed no statistically significant differences compared with women in the placebo group who did not develop the condition.

There were no statistically significant differences in the demographic characteristics between the women who developed pre-eclampsia and those who did not. The mean systolic blood pressure at 20 weeks of pregnancy in women who later developed pre-eclampsia was 106.5± 11.9 mm Hg and the mean diastolic blood pressure was 66.3±8.4 mm Hg.

None of the women who developed pre-eclampsia had proteinuria greater than 30 mg/dL at the beginning of the study, but by the end of pregnancy (38–40 weeks), mean systolic blood pressure was 127.5± 12.5 mm Hg and mean diastolic blood pressure was 88.5±8.3 mm Hg; all of these women had proteinuria greater than 30 mg/dL. The 47 women who developed pre-eclampsia had similar baseline plasma levels of CoQ10 compared with the 107 women who remained normotensive at 20 weeks of pregnancy (0.143±0.05 vs 0.136± 0.05 μmol/mmol, respectively; P=0.42) and also at 36 weeks of pregnancy (0.271±0.185 vs 0.241±0.14 μmol/mmol, respectively; P=0.27).

4. Discussion

The results of this study support the hypothesis that coenzyme Q10 (100 mg twice a day) supplementation given prophylactically from 20 weeks of pregnancy leads to a reduction in the rate of preeclampsia in women at risk for the condition.

Although the rate of pre-eclampsia in the population studied was slightly higher than reported previously, the baseline plasma concentrations of CoQ10 were similar to those reported in other populations of pregnant or nonpregnant women. The role of CoQ10 in placental function is probably related to its activity as an essential component of mitochondrial complexes I and III, in addition to its well-known antioxidant properties.

Poor placentation is an important predisposing factor for preeclampsia. The proposed “2-stage model” in which reduced placental perfusion (stage 1) leads to the maternal syndrome (stage 2) is likely to provide a simplified, yet largely accurate, description of the origin of severe early-onset pre-eclampsia, but may be less relevant for later-onset milder pre-eclampsia.

The proposed role of the placenta in the pathology of pre-eclampsia is also strongly supported by the rapid resolution of symptoms after delivery. In 3 large studies conducted with women at high risk for pre-eclampsia, high doses of vitamin C and vitamin E showed a lack of efficacy in preventing the condition, despite consistent evidence for a state of oxidative stress.

However, the absence of benefit and evidence of unfavorable outcomes in those studies cannot be extrapolated to other antioxidants, including CoQ10. These findings should not detract from the potential importance of oxidative stress in pre-eclampsia.

On the basis of these studies and the analysis of a further 7 studies, a recent Cochrane meta-analysis does not support routine antioxidant supplementation during pregnancy to reduce the risk of pre-eclampsia. However, the results of the present study will add to the debate and support the design of larger trials, particularly because of the complex bioenergetic and antioxidant function of CoQ10.

For example, a recent study by Tiano et al. revealed a positive effect of CoQ10 on endothelial function, and this could have particular importance in pre-eclampsia in which endothelial dysfunction is recognized to play a pathogenetic role.

The selection of the dosage of 100 mg CoQ10 twice a day was based on the hypothesis that during pregnancy nutrient requirements are increased.With higher doses of CoQ10 the effects might be more evident since the plasma levels of CoQ10 in women receiving supplementation did not continue to increase after 24 weeks of pregnancy.

However, the reason why women who were supplemented with CoQ10 and then developed pre-eclampsia had higher plasma levels of CoQ10 than women in the placebo group who also developed preeclampsia is not clear. The small number of women who developed pre-eclampsia and the considerable variation in the results might be confounding factors.

In conclusion, CoQ10 supplementation starting at 20 weeks of pregnancy appears to be a safe and well tolerated intervention, and resulted in a significant reduction in the rate of pre-eclampsia. More clinical studies are needed to investigate this further.

Acknowledgments

This trial was funded by Secretaria Nacional de Ciencia y Tecnología (SENACYT), Ecuador (grant PFN-053). Coenzyme Q10 and placebos were provided free of charge by Jarrow Formulas Inc.

Pretreatment with coenzyme Q10 improves ovarian response and embryo quality in low-prognosis young women with decreased ovarian reserve: a randomized controlled trial

Yangying Xu1,2,3, Victoria Nisenblat2,3, Cuiling Lu2,3, Rong Li2,3, Jie Qiao2,3, Xiumei Zhen2,3* and Shuyu Wang1*

Abstract

Background: Management of women with reduced ovarian reserve or poor ovarian response (POR) to stimulation is one of the major challenges in reproductive medicine. The primary causes of POR remain elusive and oxidative stress was proposed as one of the important contributors. It has been suggested that focus on the specific subpopulations within heterogeneous group of poor responders could assist in evaluating optimal management strategies for these patients. This study investigated the effect of anti-oxidant treatment with coenzyme Q10 (CoQ10) on ovarian response and embryo quality in young low prognosis patients with POR.

Methods: This prospective, randomized controlled study included 186 consecutive patients with POR stratified according to the POSEIDON classification group 3 (age < 35, poor ovarian reserve parameters). The participants were randomized to the CoQ10 pre-treatment for 60 days preceding IVF-ICSI cycle or no pre-treatment. The number of high quality embryos was a primary outcome measure.

Results: A total of 169 participants were evaluated (76 treated with CoQ10 and 93 controls); 17 women were excluded due to low compliance with CoQ10 administration. The baseline demographic and clinical characteristics were comparable between the groups. CoQ10 pretreatment resulted in significantly lower gonadotrophin requirements and higher peak E2 levels. Women in CoQ10 group had increased number of retrieved oocytes (4, IQR 2–5), higher fertilization rate (67.49%) and more high-quality embryos (1, IQR 0–2); p < 0.05. Significantly less women treated with CoQ10 had cancelled embryo transfer because of poor embryo development than controls (8.33% vs. 22.89%, p = 0.04) and more women from treatment group had available cryopreserved embryos (18.42% vs. 4.3%, p = 0.012).

The clinical pregnancy and live birth rates per embryo transfer and per one complete stimulation cycle
tended to be higher in CoQ10 group but did not achieve statistical significance.

Conclusion: Pretreatment with CoQ10 improves ovarian response to stimulation and embryological parameters in young women with poor ovarian reserve in IVF-ICSI cycles. Further work is required to determine whether there is an effect on clinical treatment endpoints.

Keywords: Poor ovarian response, POSEIDON stratification, Oxidative stress, Coenzyme Q10, In vitro fertilization, High-quality embryos, Clinical outcomes

Background

Poor response to controlled ovarian hyperstimulation (COH) remains one of the main challenges of the
assisted reproductive technology (ART) treatments. Despite impressive advances in the field, many women exhibit inadequate response to gonadotrophins, referred to as ‘poor or low responders’ and have higher odds of cycle cancellation, fewer oocytes at retrieval, lower oocyte quality and reduced number of embryos for transfer. Collectively, this results in serial failure of the ART cycles and is frustrating for both patients and their caregivers. The exact incidence of the condition is hard to establish owing to variable definitions in literature with the estimates ranging from 5.6 to 35.1% of ART cycles Multiple interventions have been proposed to improve reproductive outcomes in women with poor ovarian response (POR), but the randomized intervention studies and meta-analyses of these studies reveal conflicting results.

Currently, the evidence-basedtherapeutic strategies to improve ovarian response and
reproductive outcomes in women with POR are lacking, and treating clinicians often offer empirical treatments with little clinical evidence to support their use. Furthermore, it has been increasingly acknowledged that the available ovarian reserve tests are not reliable to predict pregnancy after assisted conception. We do not have universally accepted tests to predict response to treatment, which is of important value for counseling couples regarding their treatment pathways and for setting patients’ expectations.

It has been proposed that a heterogeneity of the included population is the main barrier in evaluating the
interventions and the factors that guide prognosis for POR.

An internationally-agreed consensus on the definition of POR reached by an ESHRE Campus Workshop held in Bologna in 2010 suggests that at least 2 out of 3 features must be present: advanced maternal age or any other risk factor for POR;  previous POR; abnormal ovarian reserve test. This uniform definition, however, implies that POR constitutes heterogeneous group of women with respect to age, previous reproductive experience and ovarian reserve tests that may have different response to the interventions.

While age-dependent decline in ovarian reserve and oocyte quality accounts for poor response in older women, an underlying etiology for its occurrence earlier in life is less clear. It is possible that younger women with compromised ovarian reserve represent a distinct subpopulation within POR group, and their fertility prognosis may differ from that of older women with low ovarian reserve markers or from similar age women with adequate ovarian reserve parameters but suboptimal response to ovarian stimulation.

Taking the above considerations into account, the recently established POSEIDON group (Patient-Oriented Strategies Encompassing Individualize Oocyte Number) proposed a new stratification of women with POR undergoing ART treatments, which includes 4 subgroups based on women’s age, ovarian reserve parameters and previous response to ovarian stimulation.

The POSEIDON concept introduces personalized medicine approach to the POR population and is expected to be more effective in identifying the subsets of patients who could benefit from specific interventions. The physiology of poor ovarian response is not fully understood and the molecular events underlying POR remain unknown.

Oxidative stress and mitochondrial dysfunction are among the most investigated possible mechanisms. Mitochondria are the most abundant organelles in oocytes and early embryos that generate approximately 90% of reactive oxygen species (ROS), the end products of oxygen metabolism, and convert ROS into an inactive state via antioxidant defense mechanisms.

Higher levels of ROS accumulating in mitochondria during multiple physiological conditions contribute to mitochondrial dysfunction and increase in oxidative stress. This, in turn, leads to oxidative damage to DNA and other intra-cellular aberrations, which are similar to the age-related changes.

Thus, improving mitochondrial function by supplementing antioxidants has been proposed as one of the important strategies to enhance reproductive performance.

Coenzyme Q10 (CoQ10) is a lipid-soluble coenzyme and is an essential component of the inner mitochondrial membrane. CoQ10 is primarily involved in electron transport in the mitochondrial respiratory chain and oxidative phosphorylation to produce adenosine triphosphate (ATP).

CoQ10 acts as an antioxidant by inhibiting lipid peroxidation and DNA oxidation, thus is capable of strengthening endogenous antioxidant system within a cell. CoQ10 supplementation has been shown to improve cardiovascular function and male fertility.

Reduced concentrations of CoQ10 in plasma have been associated with hypogonadism and altered levels of other steroid hormones. Decrease in CoQ10 level is commonly observed in individuals in late 30th and appears to co-occur with the age-related decline in fertility and increased rate of embryo aneuploidy, suggesting a contribution of the reduced expression of CoQ10 to ovarian ageing.

Several animal studies have demonstrated that CoQ10 protects ovarian reserve, counteracts physiological ovarian ageing by restoring mitochondrial function and increases the rate of embryo cleavage and blastocyst formation.

In the clinical setting, CoQ10 supplementation led to better response to ovulation induction and decreased odds of fetal aneuploidy in 35–43-year-old women. To date, however, no study has investigated whether CoQ10 pretreatment could improve the ART treatment outcomes in young subpopulation of poor responders in a randomized setting.

On the above evidence, this study focused on investigating the effect of CoQ10 supplementation on response to ovarian stimulation in the group of young women with diminished ovarian reserve, corresponding to the Poseidon’s stratification group 3. We hypothesized that increased oxidative stress has a prominent effect on premature decline of ovarian function in these women, which could be amenable to anti-oxidant therapy.

Methods

Study design and randomization

This was a prospective randomized controlled study, conducted at the Reproductive Medical Center of the Peking University Third Hospital, a tertiary university hospital and a center of excellence in Reproductive Medicine in China. The study is reported according to the CONSORT guidelines. The flow of the patients in this study is presented in Fig.

All the participants were randomized 1:1 to either CoQ10 treatment (study group) or no treatment (control group) followed by an ART cycle.

The randomization was performed over the period of 14 months (between June 2, 2015 and July 31, 2016) by using the computergenerated randomization codes, which were then placed in the sealed, opaque sequentially numbered envelopes by a third party (nurse practitioner) who was not directly involved in the patient management or in the randomization process.

The envelopes were handed out to the participants upon completing the informed consent. The study participants and the investigators were not blinded to the patient grouping.

The participants were followed through one completed ART cycle until all frozen embryos generated from the index cycle were used or until delivery in those who achieved pregnancy.

Study population

All consecutive women who were found to have POR and were referred to IVF-ET cycle in our institution were approached. POR was defined according to the ESHRE Bologna criteria.

The study inclusion criteria were: age < 35 years, anti-Mullerian hormone (AMH) < 1.2 ng/ml, and antral follicle count (AFC) < 5, the parameters that corresponded to a low prognosis group 3 as per the POSEIDON stratification.

Exclusion criteria were: age ≥ 35 years, history of ovarian surgery, endocrine or autoimmune disease (e.g. diabetes, thyroid disease or presence of anti-thyroid antibodies or PCOS), chromosomal abnormality, uterine malformations, more than 3 previous IVF cycles, treatment with cholesterollowering drugs, previous treatment with anti-oxidants (last 5 years) or known allergy to CoQ10 or ubiquinol (the water-soluble isoform of CoQ10).

All the participants completed the questionnaire with demographic, medical and reproductive information and underwent clinical examination, pelvic ultrasound, chromosome analysis, AMH test, reproductive endocrine profile and thyroid studies. All the included women were specifically asked about any previous treatment with anti-oxidants such as CoQ10, ubiquinol, vitamin A, vitamin E, vitamin C, beta-carotene or selenium, including the duration and time of treatment.

Treatment protocols

The intervention in the study group included oral administration of CoQ10 (GNC Holdings Inc., Pittsburg, PA, USA) 200 mg three times a day, for a period of 60 days in an open label fashion. The ART treatment (in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI)) was commenced in the first menstrual cycle upon completion of CoQ10 treatment. The control group commenced ART (IVF or ICSI) after enrollment without any additional treatment.

Ovarian stimulation and oocyte retrieval

All participants underwent ovarian stimulation with the short GnRH-antagonist protocol. A combination of recombinant follicle stimulating hormone (FSH) (Gonal-F, 225 IU/day, Merck Serono SA Aubonne Branch) and human menopausal gonadotrophin (Menotropins for injection FSH 75 IU: LH 75 IU, 225 IU/day, Livzon Pharmaceutical Group Inc.) in a fixed-dose was started on Day 2 of the menstrual cycle with the option to adjust dose according to response after 4 days of stimulation (Day 6 of menstrual cycle). GnRH antagonist (Cetrorelix 250 μg/day, Merck Serono, Darmstadt, Germany) was started when a leading follicle of 12 mm was achieved.

Recombinant human chorionic gonadotrophin (hCG) trigger (Ovidrele 250 μg; Merck Serono S.p.A, Rome, Italy) was administered when at least one follicle was above 18 mm. The cycle was cancelled when there were no follicles with diameter ≥ 14 mm after 8– 9 days of gonadotrophin therapy or when peak E2 level was below 250 pmol/l.

Ultrasound-guided transvaginal oocyte retrieval was performed 36–38 h after the trigger injection by using a 17-gauge double-lumen needle (Cook Medical) and a vacuum pump (Cook Medical) under pressure at 125 mmHg. Each follicle sized above 12–14 mm was drained, and follicle flushing was not performed. The cumulus-oocyte complexes (COCs) were removed from the collection fluid using a sterile glass pipette and washed in G-IVF Plus media (Vitrolife, Sweden) and transported to the laboratory.

Oocyte insemination and embryo culture

Oocytes were inseminated either by conventional IVF or by ICSI depending on sperm quality.Oocytes undergoing IVF insemination were placed into a dish with G-IVF (Vitrolife) covered in mineral oil.

Oocytes undergoing ICSI were denuded and injected if maturation status was confirmed by the presence of the first polar body (PB). Fertilization was assessed 17– 19 h after insemination and was defined by the presence of two pronuclears (2PN) and two PBs.

All embryos were transferred to GM medium (G-M, Life Global, CT, USA) for a further 48 h of culture.Embryo development and quality were assessed 68–72 h (day 3) after insemination, based on the number of blastomeres, blastomere symmetry, percentage of fragmentation, and quality of cytoplasm according to the criteria established by the Istanbul Consensus Workshop on Embryo Assessment . All supernumerary day-3 embryos were cryopreserved by vitrification (JIEYING laboratory Inc., Canada) for future use.

Endometrial preparation and embryo transfer

All patients underwent transfer of day-3 embryos in a fresh cycle and subsequent frozen embryo transfer (FET) when the cryopreserved embryos generated from the index stimulation cycle were available. The embryos with the best morphological grade were selected for transfer.

In absence of high-quality embryos, transfer of any embryo quality was considered after careful patient counselling. In a fresh cycle, the luteal phase was supported with progesterone intravaginal gel (Crinone 8% 90 mg/day, Merck-Serono) commenced on the day of oocyte retrieval until 14 days after embryo transfer. In women with positive pregnancy test, luteal support was continued until 8 weeks gestation.

The protocols used for FET utilized either natural cycle or artificial estradiol and progesterone endometrium priming in normo-ovulatory and oligo-ovulatory women, respectively. In natural cycle, ovulation was tracked with transvaginal ultrasound and urine LH kit. Oral dydrogesterone (Duphaston, 20 mg daily for 7 days; Abbott Biologicals B.V.) was commenced for luteal phase support 3 days after LH surge on the day of embryo transfer until 8 weeks gestation. In artificial FET protocol, oral estradiol valerate (Progynova 6 mg/day, Schering, Berlin, Germany) was initiated on the third day of the menstrual cycle and endometrial thickness was monitored with transvaginal ultrasonography.

When the endometrial thickness exceeded 8 mm, luteal support with progesterone intravaginal gel (Crinone 8% 90 mg, daily; Merck-Serono), combined with oral dydrogesterone (Duphaston, 20 mg daily for 7 days; Abbott Biologicals B.V.) was added and embryo transfer was performed after 5 days. Hormonal treatment was stopped if pregnancy test was negative or continued until 11 weeks gestation with tapering off after 10 weeks.

Single or double cleavage-stage embryo transfer were performed by using a soft catheter (K-Soft 5100; Cook, Queensland, Australia) without ultrasound guidance. Serum hCG was measured 14 days after embryo transfer and was considered positive for hCG level ≥ 10 IU. Transvaginal ultrasonography at 30 days after transfer was used to confirm clinical pregnancy.

Hormone assay procedures

All the hormonal assays were performed at the endocrine laboratory of the Peking University Third Hospital Reproductive Centre by using commercially available kits.

Serum concentrations of hCG were determined by using the commercially available ELISA kit (Beckman DXI800, Beckman, USA) according to the manufacturer’s instructions. Serum levels of anti-Mullerian hormone (AMH) were measured by automated assays using commercially available kit (Ashlab, USA).

Serum luteinizing hormone (LH), FSH, estradiol (E2), and Progesterone (P) were tested using the Immulite 1000 assay based on chemiluminescence (DPC, Poway, CA). The lower detection limit of the hCG and the AMH assays was 0.5 IU/L and 0.06 ng/ml respectively. The intra- and inter-assay coefficient of variation (CV) for hCG activity was 5% and for AMH was 8%. The lower detection limit of LH, FSH, E2 and P was 0.05 IU/L, 0.12 IU/L, 73.4 pmol/L, 0.64 nmol/L, respectively. The CV of LH and FSH was 6% and of E2 and P was 10%.

Outcome measures

The primary outcome measure was the number of high quality day-3 embryos generated from one stimulation cycle. High quality embryos were defined as embryos that reached 6 to 8-cell stage with cytoplasmic fragmentation occupying less than 10% of the embryo surface and had equal size blastomeres.

The secondary outcomes included ovarian response parameters (duration of stimulation, total dose of gonadotrophins, peak E2 level and endometrial thickness on the day of hCG trigger), embryological parameters (number of oocytes retrieved, fertilization rate, number of patients with frozen embryos and number of patients who did not achieve embryo transfer) and clinical parameters (miscarriage, clinical pregnancy and live birth rate).

Fertilization rate was defined as the number of 2PN embryos divided by the number of inseminated oocytes. Clinical pregnancy was defined as a presence of intrauterine gestational sac observed on ultrasound after 30 days of embryo transfer. Miscarriage was defined as a loss of clinical pregnancy before 24 weeks of gestation.

Live birth was defined as the birth of at least one living child, irrespective of the duration of gestation. Clinical pregnancy and live birth rate were calculated per embryo transfer cycle as number of pregnancies/ live births divided per number of women who had transfer. Cumulative pregnancy and live birth rate were defined as the number of clinical pregnancies/ live births generated from the index ART cycle following fresh or frozen embryo transfer divided by all women who received treatment.

In addition, markers of ovarian reserve, including AMH, day 3 FSH and AFC were evaluated before and after CoQ10 treatment in the participants from the intervention (study) group.

Sample size calculation
The sample size calculation for this study was based on the number of high quality embryos as primary outcome. In our center women with poor response have an average 0.6–0.8 high quality embryos per woman. To detect a difference of 50% in primary outcome measure (from 0.6–0.8 to 1.0–1.2 embryos per woman) with alpha 0.05 and power 0.80, the required sample size was estimated at 76 women in each arm. When accounted for a drop out rate of 20%, each arm required 92 women.

Statistical analysis
The Student’s t-test or Mann-Whitney U test were used for comparisons of continuous variables between the groups depending on the distribution of the data. The chi-squared test or Fisher’s exact test, where appropriate, were used for comparisons of categorical variables. Results are presented as mean ± standard deviation (SD), median and interquartile range (IQR) or as percentages. Statistical significance was set at a probability (p) value < 0.05. All statistical analyses were performed using SPSS 22.0 software (IBM Corp., Armonk, NY, USA).

Results

A total of 436 women met inclusion criteria. Of them, 186 women agreed to participate and were enrolled in the study, 93 women in each arm. Among the participants who were randomized to the intervention(CoQ10 treatment) group, 17 women were excluded from the analysis for the following reasons: one woman changed her mind to undergo ART and 16 women discontinued CoQ10 treatment due to the compliance issues.

Overall, 76 women were retained in the study group and 93 women comprised the control group. All the participants shared the features of POSEIDON group 3, i.e. low prognosis patients younger than 35 years old with poor ovarian reserve pre-stimulation parameters. Baseline characteristics were comparable between the two groups with respect to age, BMI, duration of infertility, parity, ovarian reserve tests and causes of infertility (Table 1).

Most participants were diagnosed with primary infertility and were ART treatment-naïve. In the treatment group, no local or systemic side effects related to the use of oral CoQ10 were reported. Sequential measurements of ovarian reserve markers before and after CoQ10 treatment are presented in Table 2. The levels of basal day-3 FSH were significantly lower after 60 days supplementation of CoQ10 compared to the pre-treatment levels in the same group of women. In contrast, the levels of AMH and AFC were almost identical before and after CoQ10 treatment
(Table 2).

The parameters of ovarian response to stimulation and the embryology outcomes of ART cycles in the study population are summarized in Table 3. The amount of gonadotrophin used was significantly lower in CoQ10 treatment group than in controls (p = 0.03).

The duration of gonadotrophin therapy tended to be shorter in the participants treated with CoQ10, but the difference did not reach statistical significance (p = 0.08). Peak E2 serum concentrations were significantly higher in the CoQ10 group, but there was no difference in the mean endometrial thickness on the day of hCG trigger between the two groups.

In the CoQ10 treatment group there were fewer cancelled cases due to suboptimal ovarian response (5.23%, 4/76) compared to the control group (10.75%, 10/93) although this difference failed to achieve statistical significance, p = 0.27. Overall, 94.74% (72/76) women from the CoQ10 group and 89.25% (83/ 93) women from the control group received hCG and underwent oocyte retrieval. The median number of retrieved oocytes was significantly higher after CoQ10 pretreatment (4, IQR 2–5), than in controls (2, IQR 1–2), p = 0.002.

Most women had conventional IVF and the number of ICSI cycles was comparable between the groups. The median number of fertilized oocytes and fertilization rate were significantly higher in women treated with CoQ10 than in controls, p < 0.05. The median number of high quality day 3 embryos available per patient in the CoQ10 group was 1 (IQR 0–2) and in control group was 0 (IQR 0 1.75), with significant difference in favor of CoQ10 treatment, p = 0.03.

Among the patients in CoQ10 group who underwent oocyte retrieval, there was significantly lower number of women who did not achieve embryo transfer because of failure to retrieve oocytes or due to the absence of useable embryos (8.33%, 6/72) compared to women from the control group (22.89%, 19/83), p = 0.04 (Table 4). Collectively, embryos were available for 66 women in the CoQ10 group and 64 women in the control group, all of whom underwent fresh embryo transfer.

The number of fresh embryo transfer cycles in the CoQ10 groups was comparable to that in controls. More patients in the CoQ10 group had cryopreserved embryos (18.42%, 14/ 76 vs. 4.3%, 4/93, respectively, p = 0.02) and the number of frozen-thaw embryo transfers from the index stimulation cycle was significantly higher, p = 0.01 (Table 4).

In 14.29%, 2/14 women from the CoQ10 group with available cryopreserved embryos and in 25%, 1/4 controls, embryos did not recover after thawing. One to two

 

embryos were replaced into the uterus in each transfer cycle with higher median number in the CoQ10 group (2, IQR 1–2) than in controls (1, IQR 1–2), p = 0.04.

In the CoQ10 group there were 23 clinical pregnancies following fresh embryo transfer and one additional pregnancy following frozen-thaw embryo transfer. In the control group there were 16 clinical pregnancies after fresh embryo transfer and no pregnancies after frozenthaw transfer. The were no spontaneously conceived pregnancies in either group.

Successful live birth was achieved in 22 women from the CoQ10 (21 after fresh and 1 after frozen-thaw transfer) and in 14 women from the control group. Clinical pregnancy rate and live birth rate per fresh embryo transfer cycle were 34.85% and 31.82% in women treated with CoQ10, and 25% and 21.88% in controls, respectively. The clinical estimates for frozen-thaw embryo transfer were not calculated due to the paucity of the available data. When the transfers of all embryos originating from the complete ART cycle were considered, in women treated with CoQ10 the

cumulative clinical pregnancy rate after one complete cycle was 31.58%, 24/76 and the cumulative live birth rate was 28.95%, 22/76. In the control group, the cumulative clinical pregnancy rate was 17.20%, 16/93 and the cumulative live birth rate was 15.54%, 14/93, respectively.

Miscarriage rate was 8.67% in women from the CoQ10 group and 12.5% in controls. Although women from the CoQ10 group had higher clinical pregnancy and live birth rates with lower occurrence of pregnancy loss, the difference between the treatment and control groups failed to achieve statistical significance for each of these outcomes.

Discussion

In this study we demonstrated potential benefit of CoQ10 treatment in improving ovarian response to gonadotrophin stimulation in young women with low ovarian reserve. To the best of our knowledge, this is the first study that evaluated an effect of anti-oxidant treatment in specific phenotypic subgroup of women with POR.

Our results demonstrate that pre-treatment with CoQ10 resulted in significant decrease in the total amount of gonadotrophin needed to achieve ovarian response, shorter duration of stimulation, higher peak E2 levels and the number of oocytes retrieved. CoQ10 treatment led to significant increase in fertilization rate and in the number of high quality embryos.

There was significantly lower rate of cancelled cycles because of no response to stimulation, less cancelled embryo transfers because of failed embryo development and larger number of cycles with cryopreserved embryos in the CoQ10 treated group than in controls.

The clinical pregnancy and live birth rates were higher after CoQ10 treatment then in controls, but these differences failed to achieve significance, presumably due to insufficient sample size. Taken together, our data suggest that CoQ10 administration enhances ovarian response to stimulation and improves oocyte and embryo quality.

The findings of this study are approximately in line with previous reports that linked CoQ10 with improved reproductive outcomes. Small randomized placebocontrolled study in 24 participants (10 women in CoQ10 and 14 in placebo group) have demonstrated higher peak concentration of E2, increased number of high quality cleavage embryos, and a trend towards decreased aneuploidy and higher clinical pregnancy rate after 60 days treatment with 600 mg CoQ10.

However, the study was underpowered and failed to demonstrate significant difference in clinical outcomes between the groups . Another randomized controlled study in 101 young women with PCOS demonstrated that addition of CoQ10 in a dose of 180 mg during ovulation induction with clomiphene citrate improved ovarian response in clomiphene-resistant women and resulted in higher clinical pregnancy rate.

Retrospective analysis in 797 IUI and 253 IVF cycles in women older than 36– 37 years revealed that addition of 600 mg CoQ10 to dehydroepiandrosterone (DHEA) over the period longer than 1 month resulted in lower dose of gonadotrophins and higher number of mature follicles than in women treated with DHEA alone.

The authors did not demonstrate significant difference in the embryological or clinical outcomes, and the comparisons with untreated controls were not available. The plausible effect of CoQ10 on reproductive function is attributed to its effect on the antioxidative capacity and energy production in the oocyte.

CoQ10, the only synthesized lipid soluble antioxidant in humans, is an essential component of the mitochondrial respiratory chain, serving an important antioxidant function both in mitochondria and in lipid membranes. ROS-induced DNA damage in ovary leads to genomic instability, mutations and apoptosis of oocytes, and is thought to be ameliorated by an antioxidant activity of CoQ10.

CoQ10 has been also shown to improve mitochondrial function and restore energy production by mitochondria. Mitochondrial dysfunction in oocytes results in decreased oxidative phosphorylation and suboptimal levels of mitochondria-generated ATP, which has been strongly associated with poor reproductive performance, including diminished ovarian reserve, poor oocyte quality, abnormal fertilization and deranged preimplantation embryo development.

Energy production by mitochondria is important for steroid hormone biosynthesis, oocyte maturation, fertilization, and early embryonic development. It has been demonstrated that CoQ10 supplemented in aged animal model has improved mitochondrial membrane potential, mitochondrial ATP production and mitotic spindle orientation.

Treatment with CoQ10 increased the number of ovulated oocytes and reduced ROS in oocytes to the levels observed in young animals, indicating this is an effective strategy to reverse the effect of reproductive ageing.

In humans, levels of CoQ10 in the follicular fluid positively correlated with oocyte maturation, embryo grade and pregnancy rate in women undergoing ART. While oocyte appears to be the main target of CoQ10, it remains unclear whether anti-oxidant treatment also improves uterine environment.

We did not demonstrate any differences in endometrial thickness, but there were no data to confidently comment on the effect of CoQ10 in intra-uterine milieu.

CoQ10 has been also associated with improved ovarian reserve. In rodents, CoQ10 administration reversed ovarian toxicity of cisplatin, leading to increase in the serum AMH concentrations, improved AMH-positive follicle count and lower number of atretic follicles.

Exposure to CoQ10 restored ovarian reserve in mice with induced accelerated oocyte loss. Currently, however, there is relative paucity of information concerning the exact mechanism by which CoQ10 influences ovarian reserve in humans and it is difficult to conclude whether CoQ10 rescues follicles from apoptosis or enhances primordial follicle activation.

In this study there was significant decrease in baseline FSH levels after 60 days of CoQ10 administration. It is possible that a change in FSH levels could also have occurred without CoQ10 treatment over a period of twothree months, but this seems unlikely considering that previous study in 287 infertile men showed 14% decline in FSH levels after 3 months of CoQ10 supplementation with continuing decrease throughout 12 months therapy.

In contrast, we did not observe improvement in other ovarian reserve markers, namely AMH and AFC and such discrepancy between our and animal studies could be explained by different treatment protocols and variation in physiological parameters between species. In rodents, 8–12 weeks of CoQ10 exposure corresponds to about ¼ of the life span, which is considerably longer interval in relation to a reproductive cycle when compared to analogous treatment period in humans. It has been supposed that two months exposure to CoQ10 could improve energy production in the ovary but might not be long enough to restore prolonged effect of oxidative damage.

It should be noted that it takes about three months for a primordial follicle to reach the preovulatory stage. AMH is predominantly produced upon transition from the primordial to primary follicles when they are recruited from the dormant pool and represents early stages of growth. Thus, short duration of CoQ10 administration is likely to influence late events of follicle maturation but may not be sufficient to improve follicle recruitment evidenced by AMH levels.

Indeed, the study that reported significant increase in antral follicles in CoQ10, included women who were treated with CoQ10 for an average of 8.8 ± 6.2 months. The optimal timing, duration and dose of CoQ10 supplementation remain unclear. In this study, the duration of treatment was selected arbitrary based on previous study in IVF population.

It could be argued that CoQ10 treatment implies a delay in initiation of ART cycle and thus longer pretreatment period may be less acceptable to the patients. It has been demonstrated that CoQ10 is well tolerated and safe for healthy adults at intake of up to 900 mg/day. We were guided by previous experience in selecting the dose of CoQ10, although this was rather intuitive choice.

The main strength of this study is that it focused on a specific phenotype within a broad heterogeneous group of women with POR. All the participants shared similar demographic and clinical characteristics and had comparable pre-treatment markers of ovarian activity.

In addition, we utilized an unbiased randomization process and applied the similar laboratory and clinical protocols to all the participants. The important limitation of our study was its small sample size and we were unable to detect significant differences in clinical outcomes. Live birth is an ultimate outcome of infertility treatment and is more appropriate estimate for patient counseling.

The POSEIDON group has recently suggested that the number of oocytes needed to obtain at least one euploid embryo per patient is a more practical treatment endpoint for the studies in women with POR and helps to define the short-term goals for management.

In adopting this approach, we chose the number of high quality embryos as a primary outcome measure and calculated the sample size accordingly. High drop-out rate in the study group due to CoQ10 discontinuation was additional limiting factor that should be considered in future studies. In line with the reported by others, CoQ10 administration did not cause any adverse reactions or side effects in this study, but all women who discontinued treatment reported difficulty to comply with the CoQ10 regime requiring three times a day administration.

Finally, in this study we did not evaluate the levels of oxidative stress markers before or after treatment and did not assess the influence of other lifestyle factors that may pose women at higher risk. A threshold effect of CoQ10 may vary on individual level due to interference with other environmental exposures leading to oxidative stress and this should be considered in the design of future studies.

Conclusions

In summary, pretreatment with CoQ10 increases ovarian response to stimulation and improves oocyte and embryo quality in young low prognosis patients with diminished ovarian reserve. There is a possible beneficial effect on clinical pregnancy and live birth rates, but this needs to be confirmed in larger randomized controlled studies. Further work is required to establish the optimal length, timing and dosage of treatment and to evaluate the therapeutic effect of CoQ10 supplementation in other subgroups of low prognosis women with POR.

Acknowledgements
We sincerely thank the women who participated in the study. We also thank the medical and nursing staff of the Reproductive Medical Center of Peking University Third Hospital for their assistance in patient recruitment and management.

Funding
This study was supported by National key research and development project (2016YFC1000302) and the scientific research foundation for the returned overseas Ministry of Education (A70538–3).

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Xu et al. Reproductive Biology and Endocrinology (2018) 16:29 Page 9 of 11

Authors’ contributions
YYX took part in the patient enrolment, management and follow-up, performed data analysis and prepared the first draft of the manuscript; VN contributed to study design, data analysis and preparation of the manuscript; CL was involved in embryological experiments and contributed to study design; RL and JQ contributed to study design and were involved in critical discussions; XZ contributed to the concept, design and preparation of the manuscript and was involved in patient enrolment and management; SW coordinated the research and contributed to study design and critical discussions. All authors read and approved the final manuscript.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Peking University Third Hospital (#2014091). The clinical trial registration ID on the Chinese Clinical Trial Registry is ChiCTR-IPR-17010945, http://www.chictr.org.cn/index.aspx. All the participants provided written consent to participate in the study.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.

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Author details
1. Department of Reproduction, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100026, China. 2 Reproductive Medical Center, Department of Obstetrics and Gynecology,  Peking University Third Hospital, Beijing 100123, China. 3 Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing, China.

Received: 16 February 2018 Accepted: 6 March 2018

Red blood cell folate concentrations increase more after supplementation with [6S]-5-methyltetrahydrofolate than with folic acid in women of childbearing age1– 4

Yvonne Lamers, Reinhild Prinz-Langenohl, Susanne Bra¨mswig, and Klaus Pietrzik

ABSTRACT

Background: For the primary prevention of neural tube defects (NTDs), public health authorities recommend women of childbearing age to take 400g folic acid/d 4 wk before conception and during the first trimester. The biologically active derivate [6S]-5- methyltetrahydrofolate ([6S]-5-MTHF) could be an alternative to folic acid.

Objective: We investigated the effect of supplementation with [6S]- 5-MTHF compared with that of folic acid on red blood cell folate concentration, an indicator of folate status.

Design: The study was designed as a double-blind, randomized, placebo-controlled intervention trial. Healthy women (n 144) aged 19 –33 y received 400g folic acid, the equimolar amount of [6S]-5-MTHF (416 g), 208 g [6S]-5 MTHF, or placebo as a daily supplement for 24 wk. Red blood cell and plasma folate concentrations were measured at baseline and at 4-wk intervals.

Results: The increase in red blood cell folate over time was significantly higher in the group receiving 416 g [6S]-5-MTHF/d than in the groups receiving 400g folic acid/d or 208 g [6S]-5-MTHF/d (P 0.001). No plateau was reached in red blood cell folate concentration in the 3 treatment groups during 24 wk of intervention; however, plasma folate plateaued after 12 wk.

Conclusions: We showed that administration of [6S]-5-MTHF is more effective than is folic acid supplementation at improving folate status. In addition, the study indicates that the recommended period for preconceptional folic acid supplementation should be extended to 4 wk for maximal prevention of NTDs based on folate concentrations. [6S]-5-MTHF might be an efficient and safe alternative to folic acid. Am J Clin Nutr 2006;84:156 – 61.

KEYWORDS: Red blood cell folate, 5-methyltetrahydrofolate, folic acid, preconception supplementation, neural tube defect

INTRODUCTION

Periconceptional folic acid supplementation was shown to reduce the incidence of neural tube defects (NTDs) by 72–100% (1, 2). For primary prevention, health authorities recommend that women take a supplement of 400 g folic acid/d 4 wk before conception and during the first trimester of pregnancy (3, 4). However, because most pregnancies are unplanned and only 18 – 45% of women take periconceptional supplements (5–9), some countries have implemented mandatory food fortification with folic acid (10 –12).

After folic acid was added to grain products, a decrease in the occurrence of NTDs was observed in the United States, Canada, and Chile (13–15). Countries that have not implemented mandatory folic acid fortification are concerned about the possible harm of a high intake of folic acid (eg, delaying the diagnosis of vitamin B-12 deficiency) (16).

A possible substitute for folic acid under consideration is the naturally occurring folate form [6S]-5-methyltetrahydrofolate ([6S]-5-MTHF) that is less likely to mask a vitamin B-12 deficiency (17). The condition for the usage of [6S]-5-MTHF instead of or in addition to folic acid would be to have at least equal efficacy with respect to the prevention of NTDs.

A placebocontrolled trial to assess the efficacy of [6S]-5-MTHF on the occurrence of NTDs as primary endpoint would be unethical. However, because a relation between folate status and the risk of NTDs has been assessed, a surrogate endpoint is given which is the red blood cell folate concentration.

In a case-control study conducted in Ireland, a threshold for the lowest risk of having a child born with an NTD was estimated to be a red blood cell folate concentration 906 nmol/L (18). The objective of this double-blind, randomized, placebocontrolled intervention trial was to investigate the efficacy of daily supplementation with [6S]-5-MTHF compared with folic acid in increasing red blood cell folate, an indicator of folate status and a risk marker for NTD, in healthy women of childbearing age.

The dosage of folic acid and the equimolar amount of [6S]-5-MTHF given correspond to the recommendations of periconceptional folic acid supplementation for primary NTD prevention.

Further interest was on the kinetics of red blood cell and plasma folate over this long-term trial to investigate for a possible plateau effect in folate concentrations. By including a second group that received [6S]-5-MTHF in lower amounts, we also investigated the dose-response of [6S]-5-MTHF.

EFFECT OF [6S]-5-MTHF VS FOLIC ACID ON RBC FOLATE

SUBJECTS AND METHODS

Subjects and study design
Eligible participants for the study were healthy, young women (aged between 18 and 35 y) with normal results on routine laboratory tests (hematologic pattern, blood chemistry, and thyroid markers) and an adequate vitamin B-12 status (plasma vitamin B-12  110 pmol/L).

Women were not included if they were pregnant, lactating, or planning a pregnancy within the next months. Further exclusion criteria were regular consumption of vitamin supplements that contained folic acid or food fortified with folic acid (100 g folic acid/d during the past 4 mo), medical treatment interfering with folate metabolism, and abuse of alcohol or drugs.

Women were recruited through advertisement at the University of Bonn, Germany. After screening, 144 women aged 19 –33 y were included in the study.

The intervention was a 24-wk double-blind, placebo-controlledtrial with parallel group design. Participants were randomly assigned to receive either 400 g folic acid/d, 416g [6S]-5-MTHF/d, 208g [6S]-5-MTHF/d, or placebo. Before random assignment, participants were stratified according to their genotype for the 677C3T polymorphism of the gene encoding for 5,10 methylenetetrahydrofolate reductase (MTHFR) because homozygosity for the 677C3T MTHFR polymorphism is a risk factor for an NTD-affected pregnancy (19, 20) and affects the red blood cell folate concentration (21).

During the intervention period, 1 participant withdrew because of personal reasons. After exclusion of 7 subjects with missing values (absence on blood sampling days because of vacation or illness), 136 participants were entered in the statistical analyses.

The study was approved by the Ethics Committee of the Medical Association Hamburg, Germany, and all participants gave written informed consent. The study has been described previously when presenting the homocysteine-lowering potential of the different folate supplements (22).

Supplements
Supplements were taken as a capsule, one every morning before breakfast except on the blood sampling days when the capsule was taken after venipuncture. Both subjects and investigators were blinded to the treatment.

The supplements were manufactured by PCI Services (Schorndorf, Germany) as hard gelatin capsules, each containing a blend of magnesium stearate and microcrystalline cellulose as a filler (placebo), and either 400 g (906 nmol) folic acid (Caesar&Loretz GmbH, Hilden, Germany), 416g (906 nmol) [6S]-5-MTHF calcium salt, or 208g (453 nmol) [6S]-5-MTHF calcium salt (Metafolin; Merck Eprova AG, Schaffhausen, Switzerland). Folate contents of the capsules were measured by HPLC at the beginning and at the end of the study.

The actual amounts in the capsules aimed to provide 400 g folic acid, 416 g [6S]-5-MTHF, and 208 g [6S]-5- MTHF were 393g folic acid, 408g [6S]-5-MTHF, and 208g [6S]-5-MTHF, respectively, atthe beginning ofthe study and 382 g folic acid, 412 g [6S]-5-MTHF, and 206 g [6S]-5 MTHF, respectively, at the end of the study.

Compliance with respect to the supplement intake was assessed by pill counting at weeks 8, 16, and 24.

Assessments
Fasting blood samples were collected by venipuncture at baseline and at weeks 4, 8, 12, 16, 20, and 24 of the study.

For measurement of red blood cell and plasma folate concentrations, fasting blood samples were collected into heparinized tubes. After measurement of the hematocrit, whole blood samples for red blood cell folate analysis were diluted 1:10 with 1% ascorbic acid and incubated 30 min in the dark before storage at 80 °C. The remaining whole blood of the same sample was centrifuged (2000  g for 10 min at 4 °C) and stored as plasma aliquots at 80 °C.

Folate concentrations were measured by using the microbiological assay (23). The intraassay and interassay CVs were 2.6% and 7.2% for whole blood folate and 1.3% and 5.3% for plasma folate, respectively.

For external validation, a whole blood folate standard (National Institute for Biological Standards and Control, Hertfordshire, United Kingdom) was measured at each run. To avoid between-run variation, samples from each participant were measured in one run. Red blood cell folate concentrations were calculated according to the formula:

Red blood cell = {(folate whole blood folate x 100)
–  [plasma folate x (100 – hematocrit)]}/(hematocrit)
(1)

Blood samples used to determine the health status were taken at baseline and at week 24 and were immediately analyzed by the central laboratory of the University Hospital, Bonn. Identification of the 677C3T MTHFR genotype was conducted by using the polymerase chain reaction according to the method of Frosst et al (24).

Dietary intakes were assessed by 3-d diet records administered at baseline and at weeks 8, 16, and 24. The diet records were analyzed by using EBISpro for WINDOWS (version 4; J Erhardt, University of Hohenheim, Germany).

Statistical analysis

Because the red blood cell and plasma folate concentrations were positively skewed, data were log-transformed to normalize distribution and back-transformed to geometric means with 95% CIs. For further analyses the natural logarithms of red blood cell and plasma folate were used in all statistical tests as continuous variables.

One-factor analysis of variance (ANOVA) was used to test for between-group differences with respect to baseline characteristics, dietary folate intake, and compliance.

Within-group changes in dietary folate intake were determined in each intervention group by using paired t test adjusted to multiple comparisons (P 0.05/3). Repeated-measures ANOVA was used to examine the interaction between time and intervention and to test for changes within time and intervention with respect to red blood cell and plasma folate concentrations.

Tukey’s honestly significant difference test was carried out as post hoc analysis. In case of significant interaction, the within-group comparison was carried out by using Bonferroni post hoc test adjusted for multiple comparisons (P 0.05/6).

The dose-response relation between administration of [6S]-5-MTHF and changes in red blood cell and plasma folate concentrations was tested with linear regression, including the groups that received placebo, 208 g [6S]-5-MTHF, or 416 g [6S]-5-MTHF.

Results of all statistical calculations were considered statistically significant at P 0.05. All analyses were done by using SPSS for WINDOWS (version
12; SPSS Inc, Chicago, IL)

 

RESULTS

Baseline characteristics of the 136 subjects included in statistical analyses are presented in Table 1. At baseline, the 4 intervention groups did not differ with respect to age, body mass index, red blood cell and plasma folate concentrations, and dietary folate intake.

With respect to dietary folate intake, no change was observed throughout the study period; thus, it did not differ within the groups or between the groups at baseline and at week 8, 16, or 24. The compliance with respect to supplement intake was high and did not differ significantly between the groups (P 0.05). Ninety percent of the subjects consumed 95% of the supplements, and the other 10% of subjects consumed 86 –94% of the supplements.

The mean red blood cell and plasma folate concentrations of the 3treatment groups andthe placebo group are showninFigure 1 and Figure 2. A significant interaction was observed between time and intervention in both red blood cell and plasma folate (P 0.001 for both). In red blood cell folate, the dimension of increase over time was significantly greater in the group receiving 416 g [6S]-5-MTHF/d than in the groups receiving 400 g folic acid/d or 208  g [6S]-5-MTHF/d (P 0.001 for both) and in the group receiving 400  g folic acid/d than in the group receiving 208 g [6S]-5-MTHF/d (P 0.05).

Similarly, in plasma folate, the dimension of increase over time was significantly greaterinthe group receiving 416g [6S]-5-MTHF/dthan in the groups receiving 400 g folic acid/d or 208 g [6S]-5- MTHF/d (P 0.05 and P 0.001, respectively) and in the group receiving 400  g folic acid/d group than in the group receiving 208  g [6S]-5-MTHF/d (P 0.05).

Within-group analysis showed a continuous, significant increase in red blood cell folate concentration over 24 wk of intervention, in all 3 folate groups. A plateau, defined as no further significant increase between consecutive points in time, was not observed in red blood cell folate concentration during the study period. However, in plasma folate, a plateau was reached independent of the folate form after 12 wk of supplementation.

A significant dose-dependent effect was observed on the increase of red blood cell and plasma folate over the 24-wk administration of [6S]-5-MTHF. Pearson’s correlation coefficients were 0.873 and 0.769 for changes in red blood cell and plasma folate, respectively (P 0.001 for both). Linear regression, including the groups receiving placebo, 208
g [6S]-5-MTHF or

FIGURE 1. Geometric mean red blood cell folate concentrations over time after 24 wk of supplementation with 400 g folic acid/d (Œ, n  34), 416 g [6S]-5-methyltetrahydrofolate ([6S]-5-MTHF)/d (■, n  35), 208g [6S]-5-MTHF/d (, n  33), or placebo (F, n  34). Bars represent 95% CIs.

A significant interaction was observed between time and intervention (P 0.001, repeated-measures ANOVA). The dimension of increase over time (ie, slope) was significantly greater in the group receiving 416 g [6S]-5-MTHF/d than in the groups receiving 400 g folic acid/d or 208  g [6S]-5-MTHF/d (P 0.001 for both, Tukey’s honestly significant difference test) and in the group receiving 400 g folic acid/d than in the group receiving 208 g [6S]-5-MTHF/d (P 0.05, Tukey’s honestly significant difference test). * Significantly different from the previous time point within groups, P 0.05/6 (Bonferroni post hoc test adjusted for multiple comparisons).

FIGURE 2. Geometric mean plasma folate concentrations over time after 24 wk of supplementation with 400 g folic acid/d (Œ, n  34), 416 g [6S]-5-methyltetrahydrofolate ([6S]-5-MTHF)/d (■, n  35), 208g [6S]-5-MTHF/d (, n  33), or placebo (F, n  34). Bars represent 95% CIs. A significant interaction was observed between time and intervention (P 0.001, repeated-measures ANOVA).

The dimension of increase over time (ie, slope) was significantly greater in the group receiving 416 g [6S]-5-MTHF/d than in the groups receiving 400 g folic acid/d or 208 g [6S]-5-MTHF/d (P 0.05 and P 0.001, respectively, Tukey’s honestly significant difference test) and in the group receiving 400 g folic acid/d than in the  group receiving 208 g [6S]-5-MTHF/d (P 0.05, Tukey’s honestly significant difference test). * Significantly different from the previous time point within groups, P 0.05/6 (Bonferroni post hoc test adjusted for multiple comparisons).

416 g [6S]-5-MTHF, showed that, per 100 g [6S]-5-MTHFsupplementation over 24 wk, red blood cell and plasma folate concentrations increased by 190 and 9.6 nmol/L, respectively.

DISCUSSION

This long-term intervention trial with healthy, nonpregnant women showed a higher efficacy of the biologically active folate form [6S]-5-MTHF than the equimolar amount of folic acid with respect to the increase in folate status. As an indicator of folate status which is related to the risk of NTDs (18, 25), red blood cell folate concentration was used.

Half of the amount of [6S]-5- MTHF, 208g/d, was not as efficient as 416g [6S]-5-MTHF/d or 400 g folic acid/d with respect to change over time. After 24 wk of each of the folate administrations, the mean red blood cell folate concentration exceeded 906 nmol/L, which is the concentration above which women were shown to have the lowest risk of an NTD-affected pregnancy than were case subjects with red blood cell folate concentrations  905nmol/L (18).

Daly et al (26) showed that median red blood cell folate concentration exceeded 906 nmol/L after 24 wk of supplementation with either 400 g or 200 g folic acid/d in a study of Irish women. In their study, no blood samples were drawn between baseline and week 24. Inthe present study, mean red blood cell folate concentrations exceeded 906 nmol/L after 8 wk of intervention in the groups that received 400g folic acid/d and 416g [6S]-5-MTHF/d and also after 16 wk in the group that received 208 g [6S]-5-MTHF/d.

Venn et al (27) did not observe significant differences in the increase of red blood cell or plasma folate concentration after 24 wk of supplementation with 100 g folic acid/d or the equimolar amount of [6S]-5 MTHF in a subgroup of healthy women.

In their study, the subjects’ red blood cell folate concentrations were already near 906 nmol/L at baseline. The analytic method used for folate determination was the microbiological assay both in the present study and in the studies from Daly et al (18), Daly et al (26), and Venn et al (27).

Red blood cell folate concentrations were used to estimate NTD risk by Daly et al (18) because the folate status was retrospectively measured at a median of 15 wk of gestation, whereas the time of interest was the folate status at neural tube closure (ie, at 4 wk of gestation). With respect to fetal development, the milieu providing folate to the embryo is the maternal plasma.

Low maternal plasma folate was also observed to be related to NTDs (28, 29) and other adverse pregnancy outcomes, eg, early spontaneous abortion (30). In the present study, plasma folate concentrations reached a plateau after 12 wk of folate supplementation independent of the form and dosage of folate supplementation. At this point, mean red blood cell folate concentrations had exceeded 906 nmol/L only in the groups that received 400 g folic acid/d and 416 g [6S]-5-MTHF/d. Current recommendations are for women to use periconceptional supplementation with 400 g folic acid/d 4 wk before conception and during the first trimester of pregnancy (3, 4).

Because plasma folate plateaued after 12 wk of supplementation while mean red blood cell folate concentrations had reached the “safe range,” the results of the present study show a possible additional preventive effect if women would start earlier with periconceptional folic acid and folate supplementation at amounts 400 g/d. The amount of 208 g [6S]-5-MTHF/d would be too low to reach red blood cell folate concentrations 906 nmol/L before plasma folate plateaus.

In contrast to plasma folate, red blood cell folate concentration did not reach a plateau in any of the 3 treatment groups during 24 wk of intervention. The importance of red blood cell folate in the embryonic development is that red blood cells serve as a folate storage tissue and provide folate to the maternal plasma in case of decreasing folate intake.

A plateau in red blood cell folate concentration was expected after 16 wk ( 120 d) because red blood cells incorporate folate only during erythropoieses, lose it during their degeneration, and have a mean life span of 120 d (31–33). After 120 d of folate supplementation, the new generation of red blood cells would have incorporated high amounts of supplemented folate.

However, no plateau was reached in a period of 24 wk (ie, 168 d) of folate supplementation in our study.We hypothesize that folate released during the degeneration of red blood cells is again available for incorporation in newly formed red blood cells.

Therefore, a plateau would not be achieved after the red blood cells all were replaced once (ie, 120 d); however, during the second period of 120 d, these red blood cells then would benefit from the high supply of folate through the supplements and from the folate available of the first generation of red blood cells. Folic acid supplementation was shown to decrease the mother’s risk of having an NTD-affected pregnancy (1, 2), especially when supplements are taken both before and after conception (34). Although promotion campaigns and educational programs were undertaken in the United Kingdom, Ireland, and Australia to increase the awareness and usage of periconceptional folic acid supplementation, only 18 – 45% of women were taking supplements during the recommended time span (5–9).

Prevalent characteristics of women not using folic acid supplements were unplanned pregnancy, low socioeconomic status, and late information or no knowledge about folic acid (6 – 8). With respect to the low usage of periconceptional folic acid supplementation and the high percentage of unplanned pregnancies, food fortification seems to be a more efficient strategy to increase folate status in young women andtolowertheincidence of NTDsthan education programs or campaigns for folic acid supplementation (6, 35– 37).

Food fortification has shown to decrease the occurrence of NTDs by 50% (13–15) and to increase red blood cell folate status in different populations (12, 38, 39). Countries not implementing mandatory or voluntary food fortification with folic acid generally are concerned about the possible harm of chronic exposure to high amounts of folic acid.

The possible negative side effect of the consumption of foods fortified with folic acid is, mainly in combination with the usage of vitamin supplements, that some people may exceed the tolerable upper intake level (UL) of folic acid (40, 41). Folic acid above the UL of 1 mg/d potentially can delay the appearance of the hematologic symptoms of vitamin B-12 deficiency (10, 42).

Vitamin B-12 deficiency mainly occurs in the elderly with a prevalence of 8 –16% (42), but in this age group only a small percentage exceeded the UL of folic acid (41). So far no increase in masking of vitamin B-12 deficiency was found after food fortification in the United States (43).

The possible risk for elderly people seems lower than their benefit of food fortification with respect to a decrease in plasma total homocysteine concentrations (44), an independent risk factor for vascular diseases (45).

It is more of a concern that children showed a high folic acid intake with 26% exceeding the UL (41); however, the effect of high folic acid exposure is still unknown. Unlike folic acid, [6S]-5-MTHF was proposed not to mask a vitamin B-12 deficiency according to 2 hypotheses.

First, high amounts of [6S]-5-MTHF cannot be formed into folate derivatives needed for DNA and cell synthesis if vitamin B-12 is lacking for regeneration of 5-MTHF to tetrahydrofolate (THF) (46). Second, in addition to the vitamin B-12 requirement for intracellular use of 5-MTHF, the vitamin B 12– dependent production of THF is needed for cellular retention of folate, because the preferred substrate for the folylpolyglutamate synthase is THF and it only has low affinity to 5-MTHF (47, 48).

Folic acid conversion to THF is independent of vitamin B-12; thus, it is available for intracellular use and storage. In the supplement forms used, [6S]-5-MTHF and folic acid showed equal stability in our long-term study (98.8% and 95.6%, respectively, after 6 mo). Folic acid in fortified food is stable and has a high bioavailability (49, 50).

Thus, the usage of folic acid for food fortification and [6S]-5-MTHF in vitamin supplements would be an approach to avoid the excess of the UL of folic acid. In conclusion, our study shows that administration of [6S]-5- MTHF is more effective than is folic acid supplementation at increasing red blood cell folate concentrationsin women of childbearing age. Supplementation with [6S]-5-MTHF might be an adequate alternative to folic acid for increasing folate status and, thus, for reducing the risk of having an NTD-affected pregnancy.

On the basis of red blood cell and plasma folate concentrations before conception, the recommended period of preconceptional folic acid supplementation of 4 wk should be extended to 12 wk to achieve maximal risk reduction. [6S]-5-MTHFmight beanefficientand safe alternative to folic acid in vitamin supplements.

We thank the women who participated in the study and P von Bu¨low, S Deneke, I Fohr, M Hages, R Moser, P Pickert, G Puzicha, M Schu¨ller, and O Tobolski for excellent technical assistance and valuable discussions. YL and KP had the original idea for the study and recruited the subjects.

YL, RP-L, and KP were responsible for designing and planning the study. YL was responsible for sample collection, laboratory analysis, and statistical analysis. YL, RP-L, SB, and KP contributed to the writing of the paper. None of the authors had a conflict of interest.

Folate, folic acid and 5-methyltetrahydrofolate are not the same thing

https://www.tandfonline.com/toc/ixen20/current

Francesco Scaglione and Giscardo Panzavolta
Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy

Abstract

1. Folate, an essential micronutrient, is a critical cofactor in one-carbon metabolism. Mammals cannot synthesize folate and depend on supplementation to maintain normal levels. Low folate status may be caused by low dietary intake, poor absorption of ingested folate and alteration of folate metabolism due to genetic defects or drug interactions.

2. Folate deficiency has been linked with an increased risk of neural tube defects, cardiovascular disease, cancer and cognitive dysfunction. Most countries have established recommended intakes of folate through folic acid supplements or fortified foods. External supplementation of folate may occur as folic acid, folinic acid or 5-methyltetrahydrofolate (5-MTHF).

3. Naturally occurring 5-MTHF has important advantages over synthetic folic acid – it is well absorbed even when gastrointestinal pH is altered and its bioavailability is not affected by metabolic defects. Using 5-MTHF instead of folic acid reduces the potential for masking haematological symptoms of vitamin B12 deficiency, reduces interactions with drugs that inhibit dihydrofolate reductase and overcomes metabolic defects caused by methylenetetrahydrofolate reductase polymorphism. Use of 5-MTHF also prevents the potential negative effects of unconverted folic acid in the peripheral circulation.

4. We review the evidence for the use of 5-MTHF in preventing folate deficiency.

Introduction

Folate, also known as vitamin B9, is the generic term given to a family of chemically similar compounds that have been recognized as beneficial for the prevention of a range of conditions. Folate is an essential micronutrient that is vital for normal cellular function: adequate folate intake is a critical factor in preventing some neural tube defects (NTD), has been implicated in some forms of anaemia and numerous other adverse health conditions such as cardiovascular disease and cancer (Blom & Smulders, 2011; Czeizel & Dudas, 1992; Klerk et al., 2002; Lee et al., 2011; Medical Research Council Vitamin Study Research Group, 1991; van der Put & Blom, 2000; Webb et al., 2011).

 

Plasma levels of folate are inversely related to plasma homocysteine levels at concentrations 540 mM, suggesting a link between folate intake and reduced risk of vascular disease (Forman et al., 2005; Jardine et al., 2012; Smulders & Stehouwer, 2005; Zhou et al., 2011). Furthermore, there is growing evidence that folate may play a role in the prevention of colorectal cancer, which represents the second leading cause of death due to malignancies (Sanjoaquin et al., 2005; Stolzenberg-Solomon et al., 2006). Conversely, other evidence supports a positive association between increased risk of breast cancer and high folate intake, generally attributable to supplemental folic acid rather than a diet high in folate-rich foods (Stolzenberg-Solomon et al., 2006).

 

 

This matter is still under debate. Because of the complexity of folate function, hypothetically, it is possible that both deficiency and abundance or over-supplementation of folate, in addition to other conditions, may contribute to breast carcinogenesis at different stages of tumour development or in different neoplastic or tumour phenotypes (Stolzenberg-Solomon et al., 2006). Studies of folate supplementation indicate a role in the prevention of other diseases, including neurological, cognitive and psychiatric diseases, such as cognitive dysfunction in the elderly, and in protection against degeneration of ulcerative colitis (Carrier et al., 2003; Hooshmand et al., 2012; Hwang et al., 2012; Kelly, 1998; Morris, 2012; Perez et al., 2012). Mammals, as well as all other animals, do not have the ability to synthesize folate and therefore must absorb it from the diet, however, daily dietary intake of folates is generally lower than the dosage recommended by national health authorities (Mitchell et al., 2004).

 

In fact, although natural food folates are abundant in the normal range of foods available in developed countries, many people do not eat folate-rich diets due to the cost of fresh fruit and vegetables, plus naturally occurring folates are unstable, with as much as 30% lost as a result of food processing, depending on the type of cooking used (Bergstro¨m, 1994; Bjorkegren & Svardsudd, 2003). Rich sources of folate are green, leafy vegetables, sprouts, fruits, brewer’s yeast and liver. However, a large proportion of population in lower socioeconomic groups have limited access to folate-rich foods.

 

For this reason, most countries have established increased recommended intakes of folates, introducing mandatory food fortification with synthetic folic acid (Crider et al., 2011). Although there is no a general consensus, in most countries, the recommended dietary allowance (RDA) for folate is 300 mg/day for adults and 400 mg/day for women of childbearing age. The US Food and Nutrition Board suggested a level of 400 mg/day for folic acid, expressed in terms of dietary folate equivalents (DFE) (Dietary Guidelines Advisory Committee, 2010). The introduction of folic acid-fortified primary foods has effectively decreased the prevalence of NTD (Daly et al., 1995) and stroke mortality (Yang et al., 2012).

The folate family of compounds

Folate is the generic term for a family of compounds including folic acid and its derivatives which include 5- methyltetrahydrofolate (5-MTHF), 5 formyltetrahydrofolate (5-FTHF or folinic acid), 10-formyl-THF, 5,10 methyleneTHF and unsubstituted THF. Deficiency of folate can be a direct result of low dietary intake, poor absorption of ingested folate by the intestine and increased use (i.e. physical activity, pregnancy); it can also be caused by pathological liver conditions (Halsted, 1989; Wright et al., 2005) and folate dysmetabolism, due to genetic defects or drug interactions. Folic acid is the synthetic, parent compound of this family. It is an oxidized synthetic water-soluble member of the vitamin-B complex family which does not exist in nature, although oxidation of folates to folic acid is seen in stored or cooked foods (Forssen et al., 2000). It is composed of two main units: a pteroyl group linked to a glutamic acid residue (Figure 1A). Folic acid itself is not active as a coenzyme and has to undergo several metabolic steps within the cell in order to be converted into the metabolically active THF form. In most cases, folic acid shows greater stability than the reduced folates (Forssen et al., 2000).

Folinic acid is a 5-formyl derivative of THF (Figure 1B). Unlike the synthetic folate, folinic acid is naturally found in food. It is readily converted to THF without requiring the action of the enzyme dihydrofolate reductase (DHFR).
There fore its function as a vitamin is unaffected by drugs inhibiting this enzyme, such as methotrexate (Rajagopalan et al., 2002). 5-MTHF (Figure 1C) is a biologically active form of folate and is the most abundant form found in plasma, representing >90% of folate and is the predominant active metabolite of ingested folic acid.

Folate metabolism

Folate plays an essential role in one-carbon metabolism, facilitating the transfer of one-carbon units in reactions required for the synthesis of purine and pyrimidine precursors of nucleic acids, for the metabolism of methionine, serine, glycine and histidine and for the formation of methylating agents required for normal metabolism and gene regulation (Bottiglieri et al., 1994; Lucock, 2004; Mischoulon & Fava, 2002; Reynolds, 2002; Wagner, 1995).

Folates ingested with the diet mainly exist as polyglutamates, which must behydrolysed to monoglutamates in order to be transported. This first step of folate metabolism occurs in the intestinal mucosa. Folic acid itself has no coenzyme activity until it is reduced to THF by a two-step enzymatic reaction involving a DHF intermediate and the DHFR (Blakley, 1984). THF is then metabolized by the enzyme serine hydroxymethyltransferase (SHMT) to generate glycine and 5,10-methylene-THF (Blakley, 1984; Gregory et al., 2000). 5,10-methylene-THF is in turn converted into L-5-methyl-THF [the predominant folate form found in plasma (Blom & Smulders, 2011)] by the action of the riboflavin-dependent enzyme methylenetetrahydrofolate reductase (MTHFR).

Intracellular folate metabolism is at the branch of two major inter-related metabolic cycles: synthesis of thymidylate and purines and synthesis of methionine from homocysteine (Figure 2). 5-Methyl-THF acts as a methyl donor for homocysteine remethylation which is catalysed by the vitamin B12-dependent enzyme methionine synthase.

The resulting THF can be converted into 10-formyl-THF and then into 5,10- methylene-THF by the action of the trifunctional enzyme— tetrahydrofolate dehydrogenase (MTHFD1). The 10-formylTHF serves as donor of one-carbon groups required for purine biosynthesis. THF can also be directly converted into 5,10 methylene-THF by the action of the enzyme SHMT. 5,10- Methylene-THF serves as a cofactor for the conversion of dUMP into dTMP which is catalysed by the enzyme thymidylate synthase (Blakley, 1984; Blom & Smulders, 2011). DHF, which is formed as a co-product of this reaction, is then converted to THF via DHFR.

The cycle is completed with THF accepting another one carbon unit and regenerating 5,10-methylene-THF by the action of SHMT. Within this metabolic cycle, the reaction catalysed by the enzyme MTHFR is crucial for the regulation of available 5-methylTHF, which is required for methionine synthesis. Methionine, in turn can be metabolized to S0 -adenosyl methionine (SAM), which acts as the principal methyl donor in many reactions, including methylation of DNA, histones and other proteins.

These methylation reactions play important roles in development, gene expression and genomic stability (Wolffe et al., 1999). The methionine cycle is highly sensitive to inadequate folate status (Basten et al., 2006). When folate status is poor, the ability of the cell to remethylate cellular homocysteine is impaired and this results in increased plasma homocysteine levels. Therefore, plasma homocysteine levels are an indirect indicator of folate level (Blom & Smulders, 2011).folate transport is a saturable process with a pH optimum between 5.5 and 6.0, explaining why antacids appear to reduce folate absorption (Russell et al., 1988).

Pharmacokinetics

Absorption
Dietary folate exists in the polyglutamate form, which must be converted into the monoglutamate form to be absorbed from the intestinal lumen. This reaction is catalysed by the folate conjugase such as the intestinal brush border pteroylpolyglutamate hydrolase (BB-PPH) and the intracellular hydrolase (IC-PPH) (Halsted, 1989). Intestinal absorption occurs by both passive and carrier-mediated mechanisms, with the second process predominating in the proximal small intestine. Passive absorption occurs mainly at higher doses of folate. Carrier-mediated transport occurs via three systems, namely, the reduced folate carrier (RFC), the folate receptors (FRs) and the proton-coupled folate transporter (PCFT), which transports oxidized and reduced folates with similar efficiency (Selhub et al., 1984; Sirotnak & Tolner, 1999; Subramanian et al., 2008; Zhao et al., 2009). Intestinal folate transport is a saturable process with a pH optimum between 5.5 and 6.0, explaining why antacids appear to reduce folate absorption (Russell et al., 1988).

Bioavailability
Several studies aiming at estimating the bioavailability of food folates relative to folic acid have reported values ranging between 10 and 98% (Gregory, 1995; Hannon-Fletcher et al., 2004; Tamura & Stokstad, 1973) depending on the assessment method used. Such discrepancies may be due to differences in study design, variation in the test food used, inter-subject variability, genetic and metabolic differences, lack of standardized reference methods for sample preparation and folate quantification and use of non-certified reference material (Finglas et al., 1999; Gregory, 1995; Melse-Boonstra et al., 2004; Pfeiffer et al., 2010; Summers et al., 2010; Vahteristo et al., 1996; Wright et al., 2003). In order to study folate bioavailability, both long- and short-term trials have been conducted. Long-term trials have generally focused on the analysis of folate status parameters, such as plasma folate levels, concentration of folate in red blood cells (RBC) and plasma total homocysteine. Short-term studies have evaluated the availability of folate and its active metabolites using the area-under-the-serum response-curve (AUC) method (Konings et al., 2002). Several trials report a higher relative bioavailability of supplemental folic acid compared with food folates, concluding that consumption of extra folate as natural food folate is relatively ineffective at increasing folate status (Cuskelly et al., 1996; Hannon-Fletcher et al., 2004). When external supplementation is taken into consideration, folate may be given as

Figure 3. Dose-normalized AUC of plasma[ 13C5]5-MTHF (h* nmol/L) after single oral equimolar folate doses (450 nmol ¼ 200 mg) in the form of pharmaceutical preparation with (6S)-[13C5]5-MTHF (black) or [ 13C5]folic acid (pteryol glutamic acid (PGA), black) or as bread fortified with (6S)- [ 13C5]5-MTHF (bread with MTHF, grey) or [ 13C5]folic (bread with PGA, grey). Asterisk indicates an outlier (adapted from Ohrvik & Witthoft, 2011).

folic acid or as the naturally occurring form [6S]-5-MTHF. Several studies have focused on comparing the efficacy of these two compounds in modulating folate related parameters. Lamers et al. (2006) conducted a 24-week double-blind, randomized, placebo-controlled intervention study aimed at assessing the efficacy of daily supplementation with the naturally occurring [6S]-5-MTHF compared with folic acid in increasing RBC folate in healthy women of child-bearing age.

RBC folate concentrations are useful in determining long-term folate status as they respond very slowly to changes in folate intake. This is because erythrocytes, which have a 120-day lifespan, accumulate folate only during erythropoiesis. Low serum folate is considered an indicator of folate deficiency; however, a single measurement cannot be used to differentiate between a transitory decrease in dietary folate intake and chronic deficiency states. After treatment, increases in RBC and plasma folate concentrations were significantly higher in the group receiving [6S]-5-MTHF compared with the folic acid group (Lamers et al., 2006).

The results this study support the use of [6S]-5-MTHF as an effective and safe alternative to synthetic folic acid. Fohr et al. (2002) performed an 8-week trial in which equimolar amounts of folic acid and [6R,S]-5- MTHF were administered to 160 healthy women of childbearing age.

After treatment, folate levels were measured in plasma and in RBC at time zero, and at 4 and 8 weeks. Folate plasma concentrations were significantly higher in the 5-MTHF group compared with the folic acid group, whereas the increase in RBC folate was similar in both treatment groups (Fohr et al., 2002).

Similarly, Houghton et al. (2006) conducted a 16-week trial to evaluate the effectiveness of folic acid versus [6S]-5-MTHF on RBC folate concentration during lactation. At the end of the study, the RBC folate concentration in the [6S]-5-MTHF group was higher than that in the folic acid group (Houghton et al., 2006). In short-term trials, folate availability is determined using the AUC method. A number of authors have validated this method for assessing food folate bioavailability compared with supplemental folic acid in shortterm trials (Konings et al., 2002; Prinz-Langenohl et al., 1999; Wright et al., 2005).

A combined approach of various short-term techniques has recently been reported to determine acute absorption of equimolar doses of either stable isotope-labelled [6S]-(13C5)5-MTHF or [13C5] folic acid from bread. Following the ingestion of bread fortified with [6S]-(13C5)5-MTHF the plasma AUC of this labelled folate was significantly higher than that for food labelled with folic acid. Similar results were obtained with supplemental (6S)-[13C5]5-MTHF when compared with [13C5] folic acid (Buttner et al., 2011; Ohrvik et al., 2010; Ohrvik & Witthoft, 2011) (Figure 3).

These data differ from previous works reporting no difference in short-term availability between folic acid and 5-MTHF (Pentieva et al., 2004; Prinz-Langenohl et al., 2003) and further support the influence of the methodological approach in determining folate bioavailability.

Clinical pharmacokinetic and metabolic considerations

As reported above, polyglutamate folates ingested with the diet must be converted to the monoglutamate form by the conjugase enzymes in order to be absorbed. Since these enzymes have an optimum activity at pH 6–7, alteration of the intestinal pH may determine an incomplete deconjugation of folate thus leading to reduced absorption (Halsted, 1989; Wei & Gregory, 1998). There are several conditions in which the luminal pH changes, such as atrophic gastritis and situations with altered biliary–pancreatic secretions (Russell et al., 1986).

In addition, treatment with drugs such as proton pump inhibitors (PPI) and H2 antagonists and ingestion of foods rich in citrate, malate and ascorbate may lead to alteration of luminal pH (Halsted, 1989; Russell et al., 1986). In all these conditions, supplementation with folates like folic acid is effective and generally recommended (Inskip et al., 2009; Knudsen et al., 2004).

Moreover, there are conditions in which drug treatment causes defects in folate metabolism thus impairing its conversion to the active form. This is the case for treatment with drugs such as methotrexate, aminopterine, pyrimethamine and trimethoprim which inhibit DHFR. In these conditions, folic acid supplementation is ineffective and folinic acid or 5-MTHF can be a good alternative to folic acid (Figure 4). Among the available pharmaceutical preparations, 5-MTHF shows several important advantages over folic acid. As reported above, 5-MTHF displays better performance

 

Figure 4. Genetic polymorphisms of L-5-methyltetrahydrofolate reductase (MTHFR) and implication in methotrexate depletion of systemic folate.

compared to folic acid in terms of plasma concentrations of folate (Fohr et al., 2002; Houghton et al., 2006; Lamers et al., 2006).

In addition, the reaction catalysed by DHFR, which is required to reduce folic acid to THF, is slow and easily reaches saturation. Bailey & Ayling (2009) have shown that the reduction of folic acid by DHFR per gram of human liver is on average, 52% of that in rat liver at physiological pH. Moreover, in contrast to rats, there was almost a five-fold variation of DHFR activity among the human samples. This extremely low rate of conversion of folic acid suggests that the benefit of its use in high doses will be limited by saturation of DHFR, especially in individuals possessing lower than average activity. Thus with the ever-increasing exposure to folic acid from fortification of foods and the use of supplements a total folic acid intake >1 mg is now not uncommon in USA and the low activity of DHFR in human liver is the fundamental cause of exposure to relatively high transients of plasma unmetabolized folic acid at doses greater than the RDA.

Finally, a major risk of folic acid supplementation is that it may mask vitamin B12 deficiency (Savage & Lindenbaum, 1994). In this respect, 5-MTHF would reduce this risk because, unlike folic acid, it is not able to induce a haematological response in cells from patients with vitamin B12 deficiency (Ganeshaguru & Hoffbrand, 1978; Zittoun et al., 1978).

Genetic polymorphisms of the MTHFR gene

Genetic alterations of genes codifying for key enzymes of folate metabolism may affect their activity and reduce folate availability. This would increase folate requirement and contribute to the risk of several disease conditions linked to folate status, such as NTD and cardiovascular diseases (Christensen et al., 1999; Morin et al., 2003; Rozen, 2004). In 1995, Frosst et al. observed that a thermolabile variant of MTHFR is due to a polymorphism of the MTHFR gene (677C!T polymorphism).

This mutation results in an amino acid change from alanine to valine (A222V) at a site that is critical for flavin adenine dinucleotide (FAD) binding activity and enzyme stability (Frosst et al., 1995; Wilcken et al., 2003). Such a mutation in the MTHFR gene of a developing embryo is the most established genetic risk factor for NTD and causes elevated plasma level of homocysteine (Brattstrom et al., 1998; Gudnason et al., 1998; Klerk et al., 2002).

The distribution of the polymorphism varies considerably worldwide. In the European population, up to 12% are homozygous (TT), 43% heterozygous (CT) and 45% wild-type (CC) for that polymorphism (Brattstrom et al., 1998; Gudnason et al., 1998; Klerk et al., 2002; Meleady et al., 2003).

TT homozygous frequency is lower among the African American population (1%) and higher in the Hispanic population, reaching up to 30%. In the TT genotype, the in vitro enzyme activity is reduced by 75% compared with that of the wild-type enzyme (Frosst et al., 1995; Kang et al., 1988).

The 677C !T variant of the MTHFR gene has been associated with increased risk of NTD and increased cardiovascular risk (Christensen et al., 1999; Klerk et al., 2002; Shields et al., 1999; van der Put & Blom, 2000).

In a study published by Christensen et al. (1999) the authors observed that 18–20% of the analysed sample population with NTD were homozygous for the 677C !T MTHFR polymorphism, compared to 11% for controls, implying an increased risk of NTD associated with the 677C !T polymorphism.

Another study from Shields et al. (1999) conducted on Irish population led to similar conclusions. The authors detected the homozygous TT polymorphism in 19% of NTD cases versus 8% of controls, once again supporting that the homozygous MTHFR polymorphism is an important genetic determinant in MTHFR-derived NTD risk (Shields et al., 1999).

Besides the increased risk of  developing NTD, individuals with TT homozygosis present a significantly higher cardiovascular risk due to the higher concentrations of homocysteine, especially in populations with a low dietary folate intake

 

Figure 5. Genotype and treatment: 6[S] 5-MTHF plasma concentration (ng/mL) in patients with MTHFR CC genotype or TT genotype following the
administration of 6[R,S]-5-MTHF. 6[S]-5-MTHF plasma concentration (ng/mL) in patients with MTHFR CC genotype or TT genotype following the
administration of folic acid. Reproduced with permission from Willems et al. (2004).

(Klerk et al., 2002; Rallidis et al., 2008). The methylated form of folate, N5-methyltetrahydrofolate, is required for the remethylation of homocysteine to methionine.

By inhibiting this remethylation pathway, folate deficiency induces homocysteine efflux into the circulation. Studies show a negative correlation between plasma folate, particularly N5-methyltetrahydrofolate, and circulating homocysteine levels (Durand et al., 1998).

Another polymorphism that has been studied in relation to folate metabolism and NTD is the dihydrofolate reductase (DHFR) 19-bp deletion polymorphism [a 19-bp deletion of intron 1a (DHFR19bpdel); rs70991108] (Parle-McDermott et al., 2007). The association between this polymorphism and the NTD risk was inconsistent between studies (Johnson et al., 2004; van der Linden et al., 2007).

Supplementation

External supplementation of folate may occur as folic acid, folinic acid or 5-MTHF. Supplementation of folic acid has been proven to reduce the risk of NTD and helps in reestablishing the correct levels of homocysteine in individuals with TT homozygosis (Brouwer et al., 1999; Fohr et al., 2002; Lamers et al., 2004; Venn et al., 2003).

It is now thought that naturally occurring 5-MTHF, as well as being more or at least as effective as folic acid in improving folate status, may present important advantages over synthetic folic acid and, therefore, supplementation with 5-MTHF may be a valid alternative to folic acid (Czeizel et al., 2011; Mischoulon & Fava, 2002; Obeid et al., 2013; Pietrzik et al., 2010; Reynolds, 2002; Scott, 2001).

In fact, independent studies have demonstrated that [6S]-5-MTHF displays higher bioavailability compared to folic acid, irrespective of the patient’s
genotype (Bottiglieri et al., 1994; Li et al., 2008; PrinzLangenohl et al., 2009; Willems et al., 2004) (Figure 5).

Therefore, this natural form of folate should be considered a valid alternative to folic acid supplementation or in the fortification of food products. However, specific clinical trials investigating the prevention of NTD using 5-MTHF are required. Such studies could ascertain whether the risk of NTD would be sufficiently decreased simply by increasing folate status using any folate (such as 5-MTHF) rather than specific folic acid supplementation. Daly et al. (1995) compared two approached to raise folate levels: targeting high-risk individuals or targeting the population (in which only 5% were existing users of folic acid supplements).

They found, that supplementation of high-risk women decreased the individual risk while the population approach of food fortification reduced population and suggested that the two strategies should be considered complementary in prevention of NTDs (Daly et al., 1995)

Conclusions

Low folate status is considered to be one of the most common nutritional deficiencies and although inadequate dietary intake is the primary cause genetic alterations and interactions of drugs with folate metabolism may contribute to lower folate availability.

In addition, folate deficiency may be due to low levels of vitamin B12 since this vitamin serves as a cofactor in folate metabolism. Folate deficiency has been linked to an increased risk of numerous adverse health conditions such as NTD, cardiovascular disease, cancer and cognitive disorders. External supplementation of folate may occur as folic acid, folinic acid or 5-MTHF.

Naturally occurring 5-MTHF is now known to present important advantages over synthetic folic acid. Therefore, the use of 5-MTHF instead of folic acid is strongly recommended for external supplementation and food fortification.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The authors thank Chiara Cipollina and Mary Hines for providing editorial assistance on behalf of inScience Communications, Springer Healthcare. This assistance was sponsored by Zambon.