Invited lecture delivered by Dr Sujoy Dasgupta in a Webinar by Emcure Xennex division held in June, 2021

1. Dr Sujoy Dasgupta
MBBS (Gold Medalist, Hons)
MS (OBGY- Gold Medalist)
DNB (New Delhi)
MRCOG (London)
Advanced ART Course for Clinicians (NUHS, Singapore)
Diploma, Sexual and Reproductive Medicine (South Wales, UK)
Consultant: Reproductive Medicine, Genome Fertility Centre, Kolkata
Visiting Consultant, RSV Hospital, Kolkata
Bhagirathi Neotia Women and Child Care centre
Woodlands Multispeciality Hospital, Kolkata
Managing Committee Member, Bengal Obstetric & Gynaecological Society (BOGS)
Secretary, Subfertility and Reproductive Endocrinology Committee, BOGS
Executive Committee Member, Indian Fertility Society (IFS)- West Bengal Chapter
Executive Committee Member, Indian Society for Assisted Reproduction (ISAR)- Bengal
Winner, Prof Geoffrey Chamberlain Award, RCOG World Congress, London, 2019
Role of Folic acid in prevention of birth defects
Quatrefolic –The new kid on the block

2. Fetal anomalies
• An estimated 2,95,000 newborns die within 28 days of birth
every year, worldwide, due to congenital anomalies.
• March of Dimes (MOD) Global Report on Birth Defects-94% of
these births occur in the middle and low income countries.
• Birth defects account for 7% of all neonatal mortality
• India- prevalence varies from 61 to 69.9/1000 live births.
• 70% of birth defects are preventable.
2
WHO 2020

3. Indian people ..living in the midst of risk factors for birth defects
• High fertility,
• Large number of unplanned pregnancies
• Poor coverage of antenatal care
• Poor maternal nutritional status
• High consanguineous marriages rate,
• High carrier rate for hemoglobinopathies
3
Indian J Hum Genet. 2013; 19(2): 125–129.

4. Neural Tube Defects
• Role in Prevention:
– NTD are thought to result from a dietary deficiency of
folate and/or a genetic defect affecting folate
metabolism.
– During pregnancy, the neural tube is formed from the 18th
to the 26th DAY of gestation.

5. Consequences of folate deficiency in pregnancy
• The accumulating evidence has shown that maternal folate status is associated
with increased incidence of NTD¹
• The most common birth defect in India is neural tube defects (NTDs)²
• Presentations vary from anencephaly, encephalocoele to spina bifida occulta or
cystica²
• Birth prevalence of NTDs is 4.1 cases per 1000 total births (95% CI 3.1–5.4 per
1000 total births)²
1. Gupta H, Gupta P. Neural Tube Defects and Folic Acid. Indian Pediatrics 2004; 41:577-586 .
2. Allagh KP, Shamanna BR, Murthy GVS, et al. Birth Prevalence of Neural Tube Defects and Orofacial Clefts in India: A Systematic Review and Meta-Analysis. Bhutta ZA, ed. PLoS ONE. 2015;10(3):e0118961. doi:10.1371/journal.pone.0118961.

6. 6
The history of researches on folate and neural tube defects (NTDs)
NTDs neural tube defects, WHO World Health Organization, UK the United Kingdom, US the United States, PHS Public Health Service, CDC Centers for Disease Control, FDA
Food and Drug Administration
Reprod Sci 2020 Apr;27(4):963-976.

7. • The parent of folate family compounds is folic acid which has the following basic
structure.
• Humans are incapable to synthesize it so the only source is diet.
Folic Acid
Folic Acid (Vit B9)
Pteridine + PABA + Glutamate

20. The role of folate within the body
The various form of folate function as a single-carbon donor-acceptors in a variety of biosynthetic
reactions as shown below:
(1) Synthesis of methionine. By donation of methyl group from N-5-methyl-tetrahydrofolate and
requires vitamin B12 as a coenzyme.
(2) Pyrimidine synthesis which is a rate limiting step in DNA synthesis.
(3) Purine synthesis.
(4) Conversion of serine into glycine.
(5) Histidine catabolism.

21. 21
 Maximal absorption from the
upper jejunum
Folate absorption and transport

22. Folate and folic acid
Structurally-related compounds
(as polyglutamated forms)
Naturally found in foods
Must be hydrolyzed
Unstable (food processing and storage)
FOLATES
Synthetic oxidized form of folate
First synthetized in 1945
More stable than natural form of folate
No active - Needs to be metabolized
Added in supplements and fortified
foods
FOLIC ACID

23. Folates contained in foods are unstable and susceptible to
oxidation and they lose activity during food processing,
manufacturing and storage.
Folate and Foods
Leafy Greens Asparagus Broccoli
Papaya & Oranges
Avocado Seeds & Nuts
Brussels Sprouts
Beans, Peas, Lentils
Folate and foods
Pol. J. Food Nutr. Sci. 2010, Vol. 60, No. 2, pp. 147-151

24. Daily folate requirement and storage
• Normal mixed diet may contain as much as 700 g of folate per day
• Improper food preparation can reduce this amount close to the minimum daily
requirement
• Typical body stores of folate in a normal, healthy adult are about 10mg and are
located in liver
• If dietary folate intake or intestinal absorption ceased, the body stores would
become exhausted in about 3-4 months

25. Folate deficiency : most common nutritional deficiencies
Folate deficiency reasons:
Dietary intake is inadequate. In healthy people daily intake through the
diet is considered not sufficient to cover body needs
Increased need is not matched by an increased intake
Enzyme defects involved in the metabolization
Malabsorption
Drug use that interferes with the ability of the body to use folate

26. Applications of Folate: Preconception & Pregnancy
Neural tube defects, including spina bifida and anencephaly, are the
second most common birth defects with an incidence in Italy of 0.4–
1/1,000. (De Marco et al., 2011)
Low dietary intake of folate increases the risk of birth defects,
particularly neural tube defects (NTD).
Rapidly developing cells in the embryo may suffer by lack of
adequate folate.
Failure to produce sufficient DNA and to regulate DNA function
could lead to spontaneous abortion. (George et al., 2002)
Prevention
NTD
Spontaneous
Abortion
Lactation
Breast milk folate concentrations are maintained at the
expense of maternal folate reserves.
A lactating woman would require 128 μg/day of additional
folate in order to restore her losses. (EFSA Scientific Opinion
for folate 2014)

27. Applications of Folate Homocysteinemia
Hyperhomocysteinemia
MTHFR mutations is implicated in homocysteine levels, a
risk factor for recurrent embryo losses in early pregnancy.
(Isotalo 2000)
Hyperhomocysteinemia is independent causal factor for
pregnancy complications. (Huang - 2009)
Women with hyperhomocysteinemia have increased risk to
have pregnancy outcome such as 7.7 fold risk for
preeclampsia.
(Refsum et al. 2006)

28. Role of folate
• Of all the nutrients, folic acid is particularly essential in pregnancy and pre-pregnancy
phase due to its role in synthesizing DNA¹
• Folic acid requirements are increased in pregnancy because of the rapidly dividing cells
in the fetus and elevated urinary losses²
• During pregnancy, the daily folate requirement is increased3
– by fetal demands,
– decrease in gastrointestinal absorption of folate
• Older age and obesity in conjunction with pregnancy may also increase folate needs3
1. Li K, Wahlqvist ML, Li D. Nutrition, One-Carbon Metabolism and Neural Tube Defects: A Review. Nutrients 2016, 8, 741; doi:10.3390.
2. WHO. Guideline: Daily iron and folic acid supplementation in pregnant women. Geneva, World Health Organization, 2012
3. 1. S. Bentley et al. Comparative Effectiveness of a Prenatal Medical Food to Prenatal Vitamins on Hemoglobin Levels and Adverse Outcomes: A Retrospective Analysis. Clin Ther. 2011;33:204–210.

29. Risk Groups: SOGC 2015
1. Personal positive or family history of other folate-sensitive congenital anomalies
• cardiac, limb, cleft palate, urinary tract, congenital hydrocephaly
2. Family history of NTD in a first or second-degree relative.
3. Diabetes (type I or II)
4. Medications
• Anticonvulsant medications:
• Carbamazepine, Valproic acid, Phenytoin,primidone, Phenobarbital
• Metformin
• Methotrexate
• Sulfasalazine, triamterene, trimethoprim (as in cotrimoxazole),
• Cholestyramine.
• Alcohol overuse
5. GI malabsorption conditions
• Crohn’s
• Active Celiac disease
• Gastric bypass surgery
• Advanced liver disease
• Kidney dialysis J Obstet Gynaecol Can 2015;37(6):534–549

30. SOGC Clinical Practice Guideline
• LOW risk group:
• Women or their male partner
• with no personal or family history of health risks for folic acid sensitive birth
defects.
• MODERATE risk group:
• Women with the following personal or co-morbidity scenarios (1–5) or
• Male partner with a personal scenario (1 and 2).
• HIGH risk group:
• Women or their male partners
• with a personal NTD history
J Obstet Gynaecol Can 2015;37(6):534–549

31. SOGC Clinical Practice Guideline
• The recurrence risk for a fetus with an NTD
• is shared by both mother’s and father’s personal
reproductive history, but
• only the mother is treated with the supplemental dose of
pre-conception/first trimester folic acid.
J Obstet Gynaecol Can 2015;37(6):534–549

32. continues
• Low risk group
• 0.4 mg/d
• beginning 3 months before conception
• continuing
• throughout the pregnancy and for
• 4–6 w postpartum or as long as breastfeeding continues
• Moderate risk group
• 1.0 mg/d
• beginning at least 3 months before conception
• continuing until 12 w.
• 0.4–1.0 mg/d
• From 12 w, continuing
• throughout the pregnancy, and for
• 4-6 w postpartum or as long as breastfeeding
Dosage Scheduleof Folic acid
J Obstet Gynaecol Can 2015;37(6):534–549

33. Dosage Schedule of Folic acid..contd
• High risk group
• 4.0 mg/day:
• beginning at least 3 months before conception, continuing
until 12 w.
• 0.4–1.0 mg
• From 12 w, continuing
• throughout the pregnancy, and for
• 4-6 w postpartum or as long as breast feeding continues
J Obstet Gynaecol Can 2015;37(6):534–549

35. • 400 mcg/day from at least 1 month before conception to 1st 3
months of pregnancy- All pregnant women
• High risk women- 5 mg/ day from at least 3 months before
pregnancy
• Given the relatively low prevalence in affected pregnancies,
screening for MTHFR is only likely to have a marginal effect on
NTD reduction.
• folate deficiency could have a direct effect on neural epithelium,
which, unlike most embryonic cells, expresses very high levels of
folate receptor.

36. Folic acid supplementation timing and pregnancy outcome
Outcome/time Preconception 1st T 2nd T 3rd T
NTD Sig benefit No benefit
PET Beneficial Sig benefit Beneficial
Anemia Beneficial
Fetal growth
restriction
Sig benefit Beneficial
Autism Beneficial No benefit
Future Sci OA. 2016 Jun; 2(2): FSO116

37. MTHFR
• Synthetic folic acid and dietary dihydrofolate must be converted to L-methylfolate
in a multistep process in which the MTHFR enzyme is responsible for the final
conversion to L-methylfolate.
• A common genetic variant of the MTHFR enzyme significantly reduces the amount
of available L-methylfolate and can lead to preterm delivery and birth defects
• 1/3rd of normal Indian population have deficiency of enzyme MTHFR that impairs
the conversion of supplemental folic acid to its active form, L-methylfolate1
• Upto 50% of folate related NTDs may be explained by MTHFR
polymorphism²*
1.Greenberg J. Bell S. Multivitamin Supplementation During Pregnancy: Emphasis on Folic Acid and L-Methylfolate. Rev Obstet Gynecol. 2011;4(3/4):126-127
2. Kirke et al. mpact of the MTHFR C677T polymorphism on risk of neural tube defects: case-control study. BMJ 2004;328:1535–6 .
* This study was done in Irish population.

38. Science of L-methylfolate
L Methyl Folate

39. L Methyl Folate- Advantages
• Higher bioavailability than plain folic acid
• Remains active in body for longer duration-
– Lesser first pass metabolism
– Effective distribution throughout the body,
– Better reabsorption through kidney
• Active even in presence of MTHFR polymorphism
• Does not mask Vitamin B12 deficiency

40. Masking of Vit B12 deficiency- Significance
• Megaloblastic anemia- caused by both B12 and B9 deficiency
• In severe vitamin B12 deficiency, the B12-dependent methionine
synthase enzyme is inactive, and cytosolic folate is ‘trapped’ as L-
5-methyl-THF (secondary folate deficiency)
• Other forms of folate are used in defective DNA synthesis
• high concentrations of folic acid can cause a hematological
response in patients with megaloblastic anaemia caused by
vitamin B12 deficiency, allowing the associated neurological
complications to progress (defective DNA synthesis)
• In frank Vit B12 deficiency, 5-methyl-THF would be metabolically
inert and would not be able to supply the other folate coenzymes
required for thymidylate synthesis

41. Red blood cell folate concentrations increase more after supplementation with [6S]-
5-methyltetrahydrofolate than with folic acid in women of childbearing age
• Objective:
– To investigate effect of supplementation with [6S]5-MTHF compared with that of
folic acid on red blood cell folate concentration, an indicator of folate status
• Study design:
– Double-blind, randomized, placebo-controlled intervention trial
– N= 144 (19-33 yrs)
– Received: 400μg folic acid, equimolar amount of [6S]-5-MTHF(416μg), 208μg[6S]-5-
MTHF,or placebo daily for 24 wk
• Results:
– 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. Red blood cell folate concentrations increase more after supplementation with [6S]-5-methyltetrahydrofolate than with folic acid in women of childbearing age. Am J Clin Nutr
2006;84:156–61

42. • Objective:
– To evaluate the tHcy-lowering potential of low dose FA and of MTHF with respect to the
MTHFR genotype
• Study design:
– randomized, placebo-controlled, double-blind study,
– N=160 women
– Received 400μg FA, equimolar amount of MTHF (480μg, racemic mixture), or a placebo daily
during an 8-wk treatment period.
– Blood samples were collected at baseline and at 4 and 8 wk
• Results:
– Folate plasma concentrations were significantly higher in the 5-MTHF group compared with
the folic acid group and placebo
5,10-Methylenetetrahydrofolate reductase genotype determines the plasma
homocysteine-lowering effect of supplementation with 5-methyltetrahydrofolate or
folic acid in healthy young women
Fohr et al. 5,10-Methylenetetrahydrofolate reductase genotype determines the plasma homocysteine-lowering effect of supplementation with 5-methyltetrahydrofolate or folic acid in healthy
young women. Am J Clin Nutr 2002;75:275–82.

43. 5-MTHF is the most bioavailable form
In 2009 Prinz-Langenhorf et al. compared pharmocokinetic parameters
of (6S)-5-MTHF and folic acid in women with MTHFR polymorphism
(677C→T).
* Servy et al J Assist Reprod Genet 2018.
Results
Physiological low dosage of (6S)-5-MTHF
(416 µg) increases plasma folate more
effectively than equimolar dosage of FA
(400 µg).

44. (6S)-5-Methyltetrahydrofolate is the only biological form of
folate
ACTIVE (6S)-5-MTHF
Adapted from Obeid 2013
The concentration of 5-MTHF in cord serum is
approximately 2 times higher than in maternal serum
(mean 35.8 vs. 15.6 nmol/L), suggesting that 5-MTHF
during pregnancy can provide an immediate source of
folate to be transported to the fetus
(6S)-5-MTHF is the main folate form in cord blood (means
89.4% of total folate)
Concentrations and percentage of different folate form
in maternal serum and umbilical cord serum from
neonates
Mothers Neonates
Concentrations
Mothers Neonates
Percentage

45. 5-MTHF is the metabolic active form
All the natural folates ingested
are turned into 5-methylfolate
as unique transportable form
It is immediately and
completely bioavailable
It is the only folate crossing
the blood-brain barrier
It does not mask
Vitamin B12 deficiency
It is the essential form
in which folates occur
and are stored in the
human body
(6S)-5-Methyltetrahydrofolate

46. Folate and UMFA
Several studies have reported an increase in serum of UnMetabolized
Folic Acid (UMFA) levels since the implementation of folic acid
fortification, with possible concern about its potential 'overdosing' and
adverse effects.
Acid folic 200-300 μg/daily intake. The
consumption of highest dosage of synthetic folic
acid results in absorption of unreduced folic
acid, which may interfere with folate metabolism
and generate UMFA.
✓ No UMFA accumulation
Adapted from Morris in 2010
Prevalence of detectable circulating
unmetabolized folic acid by quintile category of
estimated folic acid intake

47. Folate and UMFA
Potential toxic effects of UMFA
Reduction of natural killer cytotoxicity
Reduction of the immune systems to
capacity to kill off malignant or pre-
malignant cells
Enzymatic saturation of DHFR
Polymorphism associated with MTHFR enzymes
Uncontrolled assumption through fortified foods
✓ No UMFA accumulation
UnMetabolized Folic Acid (UMFA) content in the blood may be
amplified by:

48. Biotransformation of FA to 5-MTHF
Ref: Wilcken 2004, Jamil 2014, Yakub 2012
Rate-limiting enzyme that catalyzes the
irreversible conversion to 5-MTHF in the
methyl cycle
10% of the world’s population is affected
by homozygous (TT) MTHFR
polymorphism
Particularly common in some ethnics
groups, India (20%), southern Italy
(26%), and Mexico (32%)
Methyltetrahydrofolate
Reductase (MTHFR)
unnatural levels
of unmetabolized
folic acid (UMFA)
in the systemic
circulation.

49. Quatrefolic® doesn’t expose the body to unsafe UMFA
While the scientific community is evaluating the possible concern of
UMFA on the public health according to the food fortification, several
publications recommend to replace folic acid present in dietary
supplements, with the (6S)-5-MTHF.
Quatrefolic® supplementation does not aid to the potential
accumulation of UMFA in the blood.
Pfeiffel et al., “Unmetabolized folic acid is detected in nearly all serum samples from US children, adolescents,
and adults.” J Nutr 2015
Obeid et al., “Serum unmetabolized folic acid: the straw that broke dihydrofolate reductase's back?” J Nutr
2015
Obeid et al., “Folic acid causes higher prevalence of detectable unmetabolized folic acid in serum than B-
complex: a randomized trial.” Eur. J Nutr 2015

50. Quatrefolic® triumphes versus FA
FOLIC ACID
High bioavailability NO YES
UMFA in the blood
YES
at minimum doses > 200 µg/day
NO
Possible toxicity of UMFA YES NO
Requires Metabolism YES
NO
It is the metabolically active form
Reduction pathway affected by saturable
activity of DHFR in the liver
YES NO
Metabolization affected by enzyme
polymorphisms (DHFR)
YES NO
Metabolism affected by MTHFR
polymorphisms
YES NO
Controversy about the roles of chonic
high intake in cancer promotion
YES NO

51. Bioavailability comparison between Quatrefolic®, the (6S)-5-methyltetrahydrofolic calcium salt
and folic acid, after single oral dosing in rats.
✓ Peak plasma level about 20% higher.
New salt advantages:
improved bioavailability
PRE- CLINICAL TRIAL
Miraglia N et al. Enhanced oral bioavailability of a novel folate salt: Comparison with folic acid and a calcium folate salt in a pharmacokinetic study in rats. Minerva Ginecol 2016
New salt advantages: improved bioavailability

52. (6S)-5-methyltetrahydrofolate hematic peak produced by an equivalent dose of the indicated
folate source.
✓ Quatrefolic® has better bioavailability (+10%) than (6S)-5-methyltetrahydrofolate
calcium salt.
New salt advantages:
improved bioavailability
CLINICAL TRIAL
New salt advantages: improved bioavailability

53. High water solubility means the product may be better absorbed by mucosal cells which
may facilitate access to the blood and circulation with the potential for improved
bioavailability.
New salt advantages:
improved solubility
The choice of glucosamine as the salt for the metabolically active folate, the 5-
MTHF, is the result of a research aimed to combine the effectiveness, safety
and significant advantages of solubility compared to the previous calcium
folate generations
New salt advantages: improved solubility

54. (6S)-5-MTHF Ca Salt
(6S)-5-MTHF Ca Salt is less
bioavailable
Higher bioavailability. Peak plasma level
about 20% higher.
100 times less soluble in water than
Quatrefolic
Quatrefolic is totally soluble in water
(6S)-5-MTHF Ca Salt is a crystalline
salt
Quatrefolic® is in amorphous status,
offering a higher solubility and good
stability

55. New salt advantages:
chemical aspects
New salt advantages: chemical aspects

56. Results:
The tested product contains Quatrefolic® (400 mcg
plus B6 and B12) and the results show a significant
HCys reduction in comparison with baseline from 21.5
mcmol/L to 10.0 mcmol/L with the product containing
Quatrefolic ® and a reduction against the control
group (highly dosed folic acid - 5 mg)
The ideal HCys level (i.e. less than 10 μmol/L) was
reached in 55.8% of cases in the Quatrefolic® group,
and it was significantly higher than in controls.
The treatment appears to be safe, well tolerated
and effective in reducing HCys levels.
* Mazza et al., J Biol Regul Homeost Agents. 2016
Objectives*: Investigate the efficacy of nutraceuticals in lowering HCys serum levels
Quatrefolic® is effective
in lowering HCys
Quatrefolic is effective in lowering homocysteine

57. Safety and Compliance
Safety and tolerability of Quatrefolic®, as glucosamine salt of (6S)-5-
methyltetrahydrofolate, as well as the use in pregnancy and lactation, have been deeply
evaluated and supported by relevant number of biological and toxicological studies and
included in the New Dietary Ingredient (NDI) Notification at FDA, the Novel Food at EFSA
and the other international submissions reports.
The GRAS status, “Generally Recognized as Safe” has been achieved in 2010, by an
independent panel of recognized experts.
Safety and Compliance

58. 58