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Hyperhomocysteinemia: a risk factor in unexplained infertility

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Background: To study the role of hyperhomocysteinemia in unexplained infertility and the impact of its correction with vitamin and mineral supplementation. Methods: Total of 60 patients were included with 30 patients in case and 30 patients in control groups. Cases included patients with history of inability to conceive with frequent regular unprotected intercourse for at least 1 year. Controls included age matched parous females with at least one live birth and no history of abortions. Among the patients of unexplained infertility with hyperhomocysteinemia, homocysteine lowering agents were given and outcome studied in the form of lowering of homocysteine levels and number of conceptions. Results: The mean age was 28.1 years in study and 29.5 years in the control group .Mean level of serum homocysteine was significantly higher in study group than normal fertile women i.e.20.5µmol/l and 10.9 µmol/l respectively. Among the patients of unexplained infertility, 22(73.3%) were found to have range above the normal healthy levels. In these patients homocysteine lowering agents were given for 6 weeks and lowering of mean homocysteine levels was observed which was 10.4 µmol/l. Six (27.3%) patients conceived spontaneously during the follow up period. Conclusions: Serum homocysteine levels are inversely correlated with infertility. Homocysteine lowering agents have a favourable impact on the outcome of infertility and their use is suggested in cases of unexplained infertility associated with hyperhomocysteinemia. [Int J Reprod Contracept Obstet Gynecol 2013; 2(2.000): 165-171]
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http://dx.doi.org/10.5455/2320-1770.ijrcog20130611 Volume 2 · Issue 2 Page 165
International Journal of Reproduction, Contraception, Obstetrics and Gynecology
Dubey P et al. Int J Reprod Contracept Obstet Gynecol. 2013 Jun;2(2):165-171
www.ijrcog.org
pISSN 2320-1770 | eISSN 2320-1789
Research Article
Hyperhomocysteinemia: a risk factor in unexplained infertility
Preeti Dubey, Neena Gupta, Seema Dwivedi, Neelam Swaroop, Pavika Lal, Vandana Thawani*
INTRODUCTION
Infertility implies an apparent failure of a couple to
conceive after one year of unprotected regular
intercourse. This is based on the observation that 90% of
the normal couples achieve conception within a year.1
Infertility is primary if conception has never occurred and
secondary if the patient fails to conceive after having
achieved a previous conception. The incidence of
infertility in any community varies between 10% and
15%.2
Unexplained infertility is a term applied to an infertile
couple whose standard investigations are normal.
Therefore, the diagnosis of unexplained infertility is one
of exclusion.3 The incidence of unexplained infertility is
approximately 15-17% among the infertile couples .4 The
cause of infertility is attributed to female factors in 40-
55%, male factors in 20-30%, both male and female in
10-40% and unexplained in 10-20%.5
Hyperhomocysteinemia (HHCY) has been underlined as
an emerging risk factor for several diseases such as
arterial and/or venous thrombosis, adverse pregnancy
outcome, congenital malformations and vascular
dementia but its role in unexplained infertility is yet
under evaluation.6-11
Department of Obstetrics & Gynecology, G.S.V.M. Medical College, Kanpur-208002, U.P., India
Received: 16 February 2013
Accepted: 22 March 2013
*Correspondence:
Dr. Vandana Thawani,
E-mail: vandana.thawani@yahoo.com, vandana.thawani@gmail.com
© 2013 Dubey P et al. This is an open-access article distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
ABSTRACT
Background: To study the role of hyperhomocysteinemia in unexplained infertility and the impact of its correction
with vitamin and mineral supplementation.
Methods: Total of 60 patients were included with 30 patients in case and 30 patients in control groups. Cases
included patients with history of inability to conceive with frequent regular unprotected intercourse for at least 1 year.
Controls included age matched parous females with at least one live birth and no history of abortions. Among the
patients of unexplained infertility with hyperhomocysteinemia, homocysteine lowering agents were given and
outcome studied in the form of lowering of homocysteine levels and number of conceptions.
Results: The mean age was 28.1 years in study and 29.5 years in the control group .Mean level of serum
homocysteine was significantly higher in study group than normal fertile women i.e.20.5µmol/l and 10.9 µmol/l
respectively. Among the patients of unexplained infertility, 22(73.3%) were found to have range above the normal
healthy levels. In these patients homocysteine lowering agents were given for 6 weeks and lowering of mean
homocysteine levels was observed which was 10.4 µmol/l. Six (27.3%) patients conceived spontaneously during the
follow up period.
Conclusions: Serum homocysteine levels are inversely correlated with infertility. Homocysteine lowering agents
have a favourable impact on the outcome of infertility and their use is suggested in cases of unexplained infertility
associated with hyperhomocysteinemia.
Keywords: Hyperhomocysteinemia, Unexplained infertility, Homocysteine lowering agents, Female infertility.
DOI: 10.5455/2320-1770.ijrcog20130611
Dubey P et al. Int J Reprod Contracept Obstet Gynecol. 2013 Jun;2(2):165-171
International Journal of Reproduction, Contraception, Obstetrics and Gynecology Volume 2 · Issue 2 Page 166
S-CH2-CH2-HC NH3+
COO-
CH2-S-CH2-CH2-HC NH3+
COO-S-adenosylmethionine
methionine adenosyltransferase
S-CH2-CH2-HC NH3+
COO-
CH2-S-CH2-CH2-HC NH3+
COO-
-
adenosine
S-adenosylhomocysteine
methionine
homocysteine
CH3 (to acceptor)
adenosine
serine
cystathionine
B6
homocysteine
S-methyltransferase
glycine
betaine
N,N-dimethylglycine
cystathionine
ß-synthase
adenosine
betaine-homocysteine
S-methyltransferase
H4-folate
5-methyl
H4-folate
CH3-(B12)
HOMOCYSTEINE
METABOLISM
Figure 1: Homocysteine metabolic pathway.
Homocysteine is metabolized through two pathways:
remethylation and transsulfuration.12 Remethylation
requires folate and B12 coenzymes; transsulfuration
requires pyridoxal-5’-phosphate, the B6 coenzyme. The
remethylation pathway requires vitamin B12, folate, and
the enzyme 5, 10-methylenetetrahydrofolate reductase
(MTHFR). In kidney and liver, homocysteine is also
remethylated by the enzyme betaine homocysteine
methyltransferase (BHMT), which transfers a methyl
group to homocysteine via the demethylation of betaine
to dimethylglycine (DMG). The transulfuration pathway
requires the enzyme cystathionine-synthase (CBS) and
vitamin B6 (pyridoxal-5’- phosphate). Once formed from
cystathionine, cysteine can be utilized in protein synthesis
and glutathione (GSH) production. Active folate, known
as 5-MTHF or 5-methyltetrahydrofolate, works in concert
with vitamin B12 as a methyl-group donor in the
conversion of homocysteine back to methionine.
Normally, about 50% of homocysteine is remethylated;
the remaining homocysteine is transsulfurated to
cysteine, which requires vitamin B6 as a co-factor. This
pathway yields cysteine, which is then used by the body
to make glutathione, a powerful antioxidant that protects
cellular components against oxidative damage.
Vitamin B2 (riboflavin) and magnesium are also involved
in homocysteine metabolism. Thus a person needs
different type of vitamins B to keep homocysteine levels
low and allow for it to be properly transformed into
helpful antioxidants like glutathione. Without adequate
levels of vitamin B2, B6, B12, folate and magnesium,
dangerous levels of homocysteine may build up in the
body. Elevations in intracellular homocysteine
concentrations with corresponding increase in blood
levels can result from augmented production or reduced
metabolism. Elevation in serum levels of homocysteine
are typically caused either by genetic defects in the
enzymes involved in metabolism or by nutritional
deficiencies in vitamin cofactors. It has been observed in
literature that these nutritional deficiencies contribute to
approximately two thirds of all cases of
hyperhomocysteinemia.
Homocysteine has been shown to induce vascular
inflammation by enhancing the expression of pro-
inflammatory cytokines, such as monocyte
chemoattractant protein 1 (MCP-1), which regulates
migration and activation of monocytes/macrophages, and
interleukin 8 (IL-8), which is an important
chemoattractant for neutrophils and T-lymphocyte.13
Second, homocysteine decreases the bioavailability of
nitric oxide (NO), which is one of the major endothelium-
dependent vasodilators that is produced by the
endothelial isoform of nitric oxide synthase (eNOS).
Dubey P et al. Int J Reprod Contracept Obstet Gynecol. 2013 Jun;2(2):165-171
International Journal of Reproduction, Contraception, Obstetrics and Gynecology Volume 2 · Issue 2 Page 167
Figure 2: Cellular and molecular mechanisms of hyperhomocysteinemia-induced cell dysfunction.
This effect is mediated by an accelerated oxidative
inactivation of NO and/or eNOS or by an increase in
serum assymetric dimethylarginine, an endogenous
inhibitor of eNOS.14 Third, hyperhomocysteinemia is
found to be associated with the production of reactive
oxygen species (ROS) in endothelial and smooth muscle
cells. The mechanism of this oxidative stress relies either
on auto-oxidation of the highly reactive thiol group of
homocysteine or on the formation of intracellular
superoxide and peroxyl radicals with concomitant
inhibition of cellular antioxidant enzymes, such as
superoxide dismutase and glutathione peroxidase.15,16
Fourth, a more recent concept concerns activation of the
unfolded protein response (UPR) that is triggered when
unfolded or misfolded proteins accumulate in the
endoplasmic reticulum (ER).17 This ER stress induced
after exposure to homocysteine ,further induces the
expression of several molecular chaperones and other
stress response proteins, which are aimed at restoring
correct protein folding or retranslocating defective
proteins back to the cytosol for degradation in the
proteasomes. In case of a prolonged ER stress, the UPR
activates apoptosis by various signaling pathways.18
Homocysteine-induced endothelial apoptosis probably
also involves other mechanisms such as the classical p53
pathway.19 Furthermore, folate deficiency and genetically
determined low MTHFR activity lead to an insufficient
remethylation of homocysteine to methionine and a
decreased SAM(S-adenosyl methionine) production and
SAM/SAH (S-adenosyl homocysteine) ratio. Insufficient
availability of SAM then results in impaired methylation
reactions, with multiple consequences. Defective
methylation may lead to aberrant gene expression
resulting in abnormal fetal development and malignant
diseases.20 Finally, dietary folate deficiency and the
resulting decreased cellular synthesis of 5,10-
methyleneTHF, as well as reduced MTHFR activity lead
to an accumulation of dUMP (deoxy-uridyl
monophosphate) and thus to an excessive incorporation
of uracil into DNA, with the subsequent repair
mechanisms increasing the risk of chromosomal
breakage.21,22
Many of these mechanisms are involved to cause
hyperhomocysteinemia induced infertility. The aim of
our study was to assess homocysteine levels in females of
unexplained infertility and to study the effect of
homocysteine level lowering agents on female infertility.
METHODS
The present study was carried out in unexplained infertile
couples attending the outpatient department of Upper
India Sugar Exchange Maternity Hospital, Department of
Obstetrics & Gynecology, attached to GSVM Medical
college, Kanpur between October 2010 and September
2012.
Dubey P et al. Int J Reprod Contracept Obstet Gynecol. 2013 Jun;2(2):165-171
International Journal of Reproduction, Contraception, Obstetrics and Gynecology Volume 2 · Issue 2 Page 168
Inclusion criteria:
1) Age 20-40 years old.
2) Cases of Unexplained infertility as diagnosed by
ASRM guidelines (2006)23 for standard infertility
evaluation which includes:
a) Absence of male factor: Normal semen analysis as
assessed by WHO 2010 guidelines
b) Adequate ovulation using either a mid luteal serum
progesterone greater than 10ng/mL, urine testing
documenting the LH surge or serial transvaginal
ultrasounds to monitor the development and rupture of a
dominant ovarian follicle.
c) Normal uterine cavity and patent tubes proved by
hysterosalpingography or laparoscopy.
3) Fertile controls included women with one or more
successful pregnancies but without gestational
complications (e.g. intrauterine growth restriction,
preeclampsia, stillbirth and abruptio placentae) and
without any abortions.
Exclusion criteria:
Women with associated male factor of infertility, gross
pelvic pathology, uncontrolled chronic disease e.g.
diabetes mellitus , tuberculosis, history of chronic pelvic
inflammatory disease , previous arterial and/or venous
thrombosis or history of first degree relatives with arterial
and/or venous thrombosis before the age of 65 years were
excluded.
Interventions:
After taking consent & informing the patients about the
procedure, all patients were subjected to complete
history taking, general examination, abdominal and
pelvic examination, ultrasound examination for pelvic
pathology, hysterosalpingography to rule out defects of
the anatomical patency of the genital tract, venous blood
sampling for serum follicular stimulating hormone,
luteinizing hormone, TSH, progesterone & prolactin,
semen analysis of the patient’s husband, venous blood
sampling from patient’s antecubital vein after overnight
fasting centrifuged for 10 min at 2500 rpm and plasma
was stored till testing for serum homocysteine levels was
done.
In our study we administered folic acid 5mg, vitamin B12
500 microgram, vitamin B6 5 mg to patients with
hyperhomocysteinemia (taking 13.5 µmol/l as cut off)
and then re-assessed them for homocysteine levels after 6
weeks. The outcomes in terms of conceptions over a
period of one year were also noted. The results were
analyzed using data software like SPSS and MedCalc.
RESULTS
We compared mean serum homocysteine values among
cases and controls and found that mean serum
homocysteine levels were 17.27 ± 9.49 µmol/L in the
cases and 10.87 µmoll/L ± 4.27 µmol/L in the control
group. Unpaired t test was applied. ‘t’ value was 3.37
and degree of freedom was 58 and the result was
statistically significant ( p value = 0.001 i.e. < 0.05).
Mean difference in values observed was 6.4 with 95%
confidence interval of the mean difference between 2.60
and 10.20.
Table 1: Comparison of serum homocysteine values
among patients of unexplained infertility and fertile
patients.
Serum
homocysteine
(µmol/L)
No. of
patients
Mean
Std
dev.
t
p
Cases
30
17.27
±
9.49
3.37
0.001
Controls
30
10.87
±
4.27
Figure 3: ROC curve analysis for serum homocysteine
levels and infertility.
ROC curve (Figure 3) was applied to serum
homocysteine values in the sample and area under the
ROC curve was 0.749 which was significant. z value was
3.774 and significance of p value 0.0002 was observed.
Further sensitivity and specificity of the test was
calculated at various values and a cut off value of 13.5
was calculated at which maximum sensitivity and
specificity was observed (sensitivity 70% and specificity
76.7%).
Majority 22 (73.3%) of patients among the cases had
hyperhomocysteinemia while majority 26(86.6%) of
controls had normal homocysteine values.
Dubey P et al. Int J Reprod Contracept Obstet Gynecol. 2013 Jun;2(2):165-171
International Journal of Reproduction, Contraception, Obstetrics and Gynecology Volume 2 · Issue 2 Page 169
Table 2: Distribution of patients according to homocysteine levels.
Homocysteine levels
No. of cases
%
No. of controls
%
Patients with hyperhomocysteinemia
22
73.33
4
13.33
Patients with normal homocysteine
levels
8
26.66
26
86.66
Total
30
100
30
100
These twenty two patients (73.3%) among 30 cases, who
were found to have serum homocysteine above the
reference range value i.e.>=13.5µmol/l, were given
intervention in the form of 6 weeks therapy of
homocysteine lowering agents. Paired ‘t’ test was applied
to the samples taken before and after and tested for
homocysteine levels.
Table 3: Serum homocysteine values before and after treatment intervention.
Mean serum homocysteine levels among the 22 cases
were 20.64µmol/L ± 8.77 µmol/L before and 10.10
µmol/L ± 4.12 mol/L after the treatment. Mean serum
homocysteine lowering was 10.54µmol/L ± 6.83 µmol/L.
95% Confidence Intervals are between 7.51 13.5
µmol/L. The result was highly significant (p value <
0.001). This result showed that our intervention leads to
significant reduction in serum homocysteine values.
Six of the 22 patients conceived in the follow up period
of one year after the intervention. However, the patients
are still under follow up as the pregnancy outcome of the
patients is under study.
Table 4: Number of conceptions among the patients
treated for hyperhomocysteinemia.
No of patients with
hyperhomocysteinemia
treated
Patients
conceived
%
22
6
27.27%
DISCUSSION
Homocysteine values have been found to be elevated
significantly in patients with unexplained infertility by
various statistical analyses in our study i.e. the
independent ‘t’ test and ROC curve analysis. Findings of
our study were also supported by the preliminary work by
D’Uva et al on female reproduction. Their study revealed
raised mean homocysteine levels of 21.05±8.78
micromoles /litre in 20 women with unexplained
sterility,19.2±6.14µmol/l for patients with recurrent
pregnancy loss versus 7.85±3.31µmol/l for controls.
Their study gives an indication that infertility and
recurrent pregnancy loss are a part of the continuum of
the hyperhomocysteinemia induced adverse effects on
female reproductive system.24
In studies by Bibi et al done in Pakistani women ,mean
fasting homocysteine levels observed were significantly
higher (p=0.04) in cases (12.82±5.18 micromoles /litre)
compared with controls (9.735±1.80 micromoles/litre)
which was in concordance with our study.25
In our study among the infertile Indian females of
unexplained infertility, we found the prevalence of
hyperhomocysteinemia to be 73.3%.Refsum H et al in a
study of 100 people from Pune , India reported that 77%
had plasma homocysteine concentrations >15
micromoles/litre.26
In our study intervention in the form of folic acid,
vitamin B6, vitamin B12, and zinc lead to significant
lowering of serum homocysteine levels. Shidfar F et al in
their study concluded that folate supplementation
decreases the serum levels of homocysteine. They studied
the effect of folate supplementation versus placebo on
40 hyper-cholestrolemic subjects. Their results were
similar to that of our study. 27In studies by Chait A et al
supplementation of vitamin B6, B12 & folate rich diet
lead to significant reduction in homocysteine levels of
studied group.28
CONCLUSIONS
Serum homocysteine values were found to be
significantly higher in the infertile females (17.29±9.49
micromoles/litre) as compared to their fertile controls
(10.87±4.27 micromoles/litre). A critical level of 13.5
µmol/l is suggested as the cut off for intervention by this
study.
Higher than usual prevalence of homocystinemia in this
cohort suggests the increased prevalence of micronutrient
deficiency due to inadequate diet or dietary fads in the
population. Micronutrient supplementation in the form of
Vitamin B6, B12 and folic acid is helpful in reducing
homocysteine values in patients with
Mean (n=22)
Standard Deviation
Paired ‘t’
p value
Before treatment
20.64
+ 8.77
7.24
0.0001
After treatment
10.10
+ 4.12
Dubey P et al. Int J Reprod Contracept Obstet Gynecol. 2013 Jun;2(2):165-171
International Journal of Reproduction, Contraception, Obstetrics and Gynecology Volume 2 · Issue 2 Page 170
hyperhomocysteinemia. However, large scale studies are
required to establish the role of hyperhomocysteinemia in
unexplained infertility.
Funding: No funding sources
Competing interests: None declared
Ethical approval: Not required
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Dubey P et al. Int J Reprod Contracept Obstet Gynecol. 2013 Jun;2(2):165-171
International Journal of Reproduction, Contraception, Obstetrics and Gynecology Volume 2 · Issue 2 Page 171
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7.
DOI: 10.5455/2320-1770.ijrcog20130611
Cite this article as: Dubey P, Gupta N, Dwivedi S,
Swaroop N, Lal P, Thawani V.
Hyperhomocysteinemia: a risk factor in
unexplained infertility. Int J Reprod Contracept
Obstet Gynecol 2013;2:165-71.
... Infertility is defined as inability to obtain fertility after one year of unprotected regular intercourse. [1][2][3][4] It has been reported that about 10% -15% of young couples suffer from infertility. Of these, 40%-55% are due to female factors, 20%-30% are due to male factors, and 15%-17% are unexplained infertility. 1 Over the years, ART (assisted reproductive technology) has been a method for infertility treatment with different causes. ...
... Of these, 40%-55% are due to female factors, 20%-30% are due to male factors, and 15%-17% are unexplained infertility. 1 Over the years, ART (assisted reproductive technology) has been a method for infertility treatment with different causes. 5,6 In order to increase the success rate of fertilization, several herbal and chemical supplemental factors have been used. ...
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Elevated blood homocysteine is a risk factor for cardiovascular disease. A 5-micromol/L increase is associated with an approximately 70% increase in relative risk of cardiovascular disease in adults. For patients with established risk factors, this risk is likely even greater. Effects of increased dietary folate and recommended intakes of vitamins B-12 and B-6 on serum total homocysteine (tHcy) were assessed in individuals at high risk of cardiovascular disease. This trial was conducted at 10 medical research centers in the United States and Canada and included 491 adults with hypertension, dyslipidemia, type 2 diabetes, or a combination thereof. Participants were randomly assigned to follow a prepared meal plan (PMP; n = 244) or a self-selected diet (SSD; n = 247) for 10 wk, which were matched for macronutrient content. The PMP was fortified to provide >/=100% of the recommended dietary allowances for 23 micronutrients, including folate. Mean folate intakes at 10 wk were 601 +/- 143 microgram/d with the PMP and 270 +/- 107 microgram/d with the SSD. With the PMP, serum tHcy concentrations fell from 10.8 +/- 5.8 to 9.3 +/- 4.9 micromol/L (P < 0.0001) between weeks 0 and 10 and the change was associated with increased intakes of folate, vitamin B-12, and vitamin B-6 and with increased serum and red blood cell folate and serum vitamin B-12 concentrations. tHcy concentrations did not change significantly with the SSD. The PMP resulted in increased intakes and serum concentrations of folate and vitamin B-12. These changes were associated with reduced serum tHcy concentrations in persons at high risk of cardiovascular disease.