Content uploaded by Ying C Cheong
Author content
All content in this area was uploaded by Ying C Cheong on Jan 09, 2016
Content may be subject to copyright.
Available via license: CC BY 4.0
Content may be subject to copyright.
Content uploaded by Nick Macklon
Author content
All content in this area was uploaded by Nick Macklon on Apr 05, 2015
Content may be subject to copyright.
ORIGINAL ARTICLE Reproductive biology
Amino acid composition of human
uterine fluid: association with age,
lifestyle and gynaecological pathology
Alexandra J. Kermack1,2,3, 4, Sarah Finn-Sell1,2, Ying C. Cheong2, 3,
Nicholas Brook3, Judith J. Eckert1, 2, Nick S. Macklon2,3,4,
and Franchesca D. Houghton1, 2,*
1
Centre for Human Development, Stem Cells & Regeneration, University of Southampton, Southampton SO16 6YD, UK
2
Academic Unit of
Human Development & Health, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK
3
Complete Fertility Centre,
Department of Obstetrics & Gynaecology, Princess Anne Hospital, Southampton SO16 6YD, UK
4
NIHR BRC in Nutrition Southampton,
Southampton SO16 6YD, UK
*Correspondence address. Centre for Human Development, Stem Cells & Regeneration, Faculty of Medicine, University of Southampton,
Duthie Building (MP808), Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK. Tel: +44-23-8120-8731;
Fax: +44-23-8120-4264; E-mail: F.D.Houghton@soton.ac.uk
Submitted on July 10, 2014; resubmitted on December 8, 2014; accepted on January 6, 2015
study question: Do the amino acid levels of human uterine fluid vary with age, BMI, phase of menstrual cycle, benign pathology or diet?
summary answer: The levels of 18 amino acids in human uterine fluid were shown to be affected only by maternal diet.
what is known already: Murine, bovine and ovine uterine amino acid content has been reported, but no reliable data on the human
exist. Murine studies have demonstrated that the intrauterine periconceptional nutritional environment is affected by maternal diet.
study design, size, duration: Uterine secretions were aspirated from 56 women aged 18– 45 years. The women were recruited
preoperatively from gynaecological theatre operating schedules or hysterosalpingo-contrast-sonography (HyCoSy) lists. A proportion of these
women had proven fertility; however, the majority were being investigated for subfertility. The BMI, gynaecological history and dietary pattern of
these women were also assessed.
participants/materials, setting, methods: Reverse phase high performance liquid chromatography was used to analyse
the concentrations of 18 amino acids within the uterine fluid and blood serum. The results were analysed against the women’s stage of cycle, age,
BMI and diet.
main results and the role of chance: The profile of 18 amino acids in uterine fluid was described. In total, human uterine fluid
was observed to contain an amino acid concentration of 3.54 mM (interquartile range: 2.27 – 6.24 mM). The relative concentrations of 18 amino
acids were not significantly altered by age, BMI, cycle phase or the presence of specific benign gynaecological pathologies. However, a diet iden-
tified by a validated scoring systemas being less healthy was associated with higher concentrations of asparagine (P¼0.018), histidine (P¼0.011),
serine (P¼0.033), glutamine (P¼0.049), valine (P¼0.025), phenylalanine (P¼0.019), isoleucine (P¼0.025) and leucine (P¼0.043) in the
uterine fluid compared with a healthier diet, defined as one with a higher intake of fresh vegetables, fruit, whole-grain products and fish and a low
intake of red and processed meat and high fat dairy products. There were no significant correlations between serum amino acid concentrations
and those in the uterine fluid.
limitations, reasons for caution: Our results enabled us to detect the effect of diet on the concentrations of amino acids in
human uterine fluid; however, the study may not have had sufficient numbers to detect mild effects of BMI or age.
wider implications of the findings: These findings increase our understanding of the nutritional environment encountered by
the preimplantation embryo, and indicate how periconceptional diet may alter this. Given the importance of early embryo environment for pro-
gramming of development and future health, this information may aid in the development of nutritional interventions aimed at optimizing the pre-
implantation phase of human embryo development in vivo.
&The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse,
distribution, and reproduction in any medium, provided the original work is properly cited.
Human Reproduction, Vol.30, No.4 pp. 917– 924, 2015
Advanced Access publication on February 18, 2015 doi:10.1093/humrep/dev008
at University of Southampton on January 9, 2016http://humrep.oxfordjournals.org/Downloaded from
study funding/competing interest(s): This work was funded by the NIHR, the Medical Research Council (G0701153) and
the University of Southampton and was supported by the NIHR BRC in Nutrition and Southampton University NHS Foundation Trust. The
authors declare no conflicts of interest.
Key words: amino acids / human uterine fluid / BMI / diet / menstrual cycle
Introduction
The mammalian embryo has been shown to be able to detect and
respond to the intrauterine environment it encounters. A period of de-
velopmental plasticity enables the embryo, and later the fetus to alter
intrauterine ‘programming’ for later life, as first enunciated by Barker
(Barker, 2004). In recent years it has become clear that maternal diet
in early pregnancy may also impact on the risk of development of
chronic diseases in later life (Barker, 2007); indeed even the environment
in which the preimplantation embryo develops has been shown to have
significant implications for development and health in later life (Dumoulin
et al., 2010;Eskild et al., 2013). Murine studies have demonstrated that a
periconceptional low protein diet affects the composition of uterine fluid,
and induces a remarkable response in preimplantation embryos,
whereby they ‘adapt’ to the low protein environment by increasing endo-
cytosis and trophoblast invasion (Watkins et al., 2008;Eckert et al., 2012;
Sun et al., 2014). While uterine fluid represents the preimplantation
milieu of the embryo, the nutritional contents of this fluid have not
been fully characterized in the human and it remains unclear how
these may be altered by factors such as female age, lifestyle and
disease. It is recognized that women with an increased age and BMI
are more likely to experience subfertility linked to obesity and polycystic
ovarian syndrome (PCOS). However, the impact of maternal diet, body
composition and age on the metabolic environment of human uterine
fluid has not been investigated.
The potential influence of diet on the nutrient composition of uterine
secretions has been demonstrated in rats and mice fed a low protein diet
during the preimplantation period, showing that diet can shape blasto-
cyst lineage differentiation. In both species there were reduced concen-
trations of amino acids in the maternal serum (Kwong et al., 2000;Eckert
et al., 2012). In rats, this resulted in blastocysts containing a reduced
number of inner cell mass (ICM) and trophectoderm (TE) cells which
led to abnormal programming of growth (Kwong et al., 2000). In
mouse models, a low protein diet fed during the preimplantation
period resulted in a reduction in branched chain amino acids in the
uterine fluid but in comparison, there were only minimal changes in
the amino acid content of the blastocyst (Eckert et al., 2012). These
data suggest that, at least in animal models, maternal diet can affect the
amino acid environment in which preimplantation embryos develop
and may have implications for improving the treatment of infertility, par-
ticularly in cases where there is no known cause.
Amino acids have a number of physiological roles during preimplanta-
tion development (Houghton, 2013). They may be used as a source of
energy (Lane and Gardner, 1998); in the synthesis of proteins and
nucleotides (Alexiou and Leese, 1992); as pH regulators (Edwards
et al., 1998), antioxidants (Dawson et al., 1998) and osmolytes
(Nasr-Esfahani et al., 1992) and as cell signalling molecules (Manser
et al., 2004). It has also been demonstrated that amino acids, particularly
leucine, have a vital role in the production and regulation of mTOR
(mammalian target of rapamycin), a serine/threonine protein kinase
which has a crucial role in cell growth and differentiation (Chen et al.,
2009).
In contrast to the human, the concentration of amino acids found in the
murine (Harris et al., 2005), ovine (Gao et al., 2009) and bovine (Fahning
et al., 1967;Shorgan, 2003;Hugentobler et al., 2007) uterine fluids has
been described. In the mouse, the uterine fluid contained a total
amino acid concentration of 7.18 +0.73 mM, which was lower than
that observed in the oviduct (Harris et al., 2005). Gao et al. examined
the changes in amino acid concentrations throughout the menstrual
cycle of ewes and found alterations in the levels of asparagine, tyrosine,
tryptophan, methionine and valine between Days 3 and 16 of the cycle
(Gao et al., 2009). Moreover, an increase in essential amino acids was
observed in the uterine fluid of pregnant as opposed to non-pregnant
heifers (Groebner et al., 2011). These variations in amino acid concentra-
tion with cycle stage and pregnancysuggest that, at least in animal models,
levels are regulated.
The inclusion of amino acids in preimplantation embryo culture
medium has been shown to be beneficial. In the mouse, non-essential
amino acids improve initial cleavage of the embryo whilst the presence
of a full complement of amino acids was beneficial for development
from the 8-cell to the blastocyst stage (Lane and Gardner, 1997). In
the human, embryos cultured in the presence of amino acids produced
blastocysts with a greater cell number in both the trophectoderm (TE)
and inner cell mass (ICM) (Devreker et al., 2001). Moreover, the
ability to use amino acid utilization to predict the future developmental
competency of individual human embryos to the blastocyst stage
(Houghton et al., 2002;Stokes et al., 2007), as well as to live birth follow-
ing transfer (Brison et al., 2004), highlights the importance of this nutrient
source. These data are intriguing and implicate amino acids as being
central for embryo development. It is therefore surprising that the full
composition of amino acids in human uterine fluid remains unknown.
In this study we sought to determine the concentration of amino acids
in human uterine fluid and how sensitive the amino acid profile is to
various factors such as female age, stage of the cycle, reproductive path-
ology, BMI and diet.
Materials and Methods
Ethical approval for this study was granted by the Southampton and South
West Hampshire Research Ethics Committee (08/H0502/162) and the Uni-
versity Hospital Southampton Research and Development department.
A total of 68 women aged 18 – 45 years were recruited to the study from op-
erating theatre schedules and hysterosalpingo-contrast-sonography
(HyCoSy) lists. Exclusion criteria included contraceptive use; current or pre-
vious history of malignancy; and known infections (including systemic).
Women were recruited preoperatively and gave their written informed
consent. Data on the women’s demographic, obstetric and gynaecological
history, BMI and diet were collected on a standard study proforma on admis-
sion. Women taking part in the study were asked about their last menstrual
918 Kermack et al.
at University of Southampton on January 9, 2016http://humrep.oxfordjournals.org/Downloaded from
period and length of cycle and from this their stage of cycle was calculated.
The women were all ovulating naturally and not undergoing ovarian induction
or stimulation. Pathologies were diagnosed by clinical history and examin-
ation, laboratory and, ultrasound assessments and when appropriate, lapar-
oscopy. Polycystic ovarian syndrome (PCOS) was diagnosed according to the
Rotterdam criteria; leiomyomas and ovarian cysts were diagnosed at ultra-
sound; and endometriosis and hydrosalpinges were diagnosed at laparos-
copy. Diet quality of the women was assessed using a validated food
frequency questionnaire (Crozier et al., 2010) to determine their compliance
with a ‘prudent’ dietary pattern. This questionnaire has been fully described
elsewhere but, briefly, a prudent diet describes a diet characterizedby higher
intakes of fresh vegetables, fruit, whole-grain products and fish and lower
intakes of red and processed meat and high fat dairy products. Depending
on the frequency of intake of key dietary elements, a score can be calculated
(Crozier et al., 2010). A positive prudent diet score was considered to indi-
cate a ‘healthy’ diet and a negative score to indicate an ‘unhealthy’ diet.
Samples of uterine fluid were obtained after the cervix was cleansed during
a speculum examination. This was done prior to commencing the planned
surgical or HyCoSy procedure by inserting an embryo transfer catheter
(Cook Medical Sydney embryo transfer catheter, USA) gently into the
uterine cavity and applying gentle suction with a 2 ml syringe, as previously
described (Boomsma et al., 2009). The catheter containing the uterine
fluid was placed in a sterile tube and snap frozen in liquid nitrogen, before
being stored at 2808C. Sixteen of the women underwent venepuncture,
and five of these were fasted prior to the blood test. The amino acid
content was determined by reverse phase high pressure liquid chromatog-
raphy (HPLC).
The uterine fluid samples were removed from the embryo transfer tubing
and diluted 1 in 10 in PBS containing 0.1% sodium dodecyl sulphate (Fisher
Scientific). Any samples which were heavily blood stained or had a volume
,10 ml were discarded. Blood samples were obtained and allowed to clot
for 30 min at room temperature. The samples were centrifuged at 2000g
for 10 min at 48C and the serum supernatant collected and stored at
2808C. Serum samples were diluted 1:1 with HPLC grade water prior to
analysis. The concentration of amino acids in the uterine fluid and the
serum were analysed using reverse phase HPLC (Agilent 1100) and calcu-
lated relative to a known concentration of amino acids. Pre-column derivati-
zation was achieved via the automated reaction of 10ml sample and 10 ml
o-phthaldialdehyde (Sigma) reagent containing 0.2% b2-mercaptoethanol
(Sigma). Amino acids were eluted using an elution gradient. Buffer A com-
prised 15 ml tetrahydrofuran (Fisher Scientific), 200 ml HPLC grade metha-
nol and 800 ml sodium acetate (83 mM, pH 5.9) and buffer B 200 ml sodium
acetate (83 mM, pH 5.9; Fisher Scientific) and 800 ml HPLC grade methanol
(Christensen et al., 2014). This method allowed the separation and analysis of
18 amino acids; including essential amino acids; histidine (His), glutamine
(Gln), arginine (Arg), threonine (Thr), tyrosine (Tyr), methionine (Met),
valine (Val), tryptophan (Trp), phenylalanine (Phe), isoleucine (Iso), leucine
(Leu), and lysine (Lys); and non-essential amino acids; aspartic acid (Asp),
glutamate (Glu), asparagine (Asn), serine (Ser), glycine (Gly), and alanine
(Ala). This method did not allow the measurement of proline and cysteine.
The amino acid concentration in the uterine fluid was measured for
women being treated for subfertility (trying to conceive without success
for at least 1 year) (n¼51) and those with normal fertility ( fertile controls,
........................................................................................................................................................
.............................................................................................................................................................................................
Table I Concentration of amino acids in human uterine fluid and serum.
Amino acid Concentration (mM)
Uterine fluid median
(n556)
Lower
quartile
Upper
quartile
Serum mean
(n516)
Spearman’s rank
correlation
Asp 0.113 0.057 0.199 0.004 +0.001 20.0312
Glu 1.189 0.636 2.059 0.028 +0.004 20.1619
Asn 0.041 0.022 0.062 0.049 +0.004 20.3486
His 0.055 0.029 0.082 0.085 +0.006 20.1047
Ser 0.142 0.072 0.256 0.117 +0.009 0.0103
Gln 0.130 0.070 0.223 0.543 +0.056 20.4348
Arg 0.190 0.070 0.314 0.097 +0.009 20.2655
Gly 0.462 0.295 0.953 0.265 +0.027 20.2979
Thr 0.192 0.108 0.310 0.126 +0.010 20.3309
Ala 0.256 0.151 0.530 0.379 +0.038 20.4874
Tyr 0.057 0.040 0.121 0.062 +0.006 20.2322
Met 0.023 0.007 0.049 0.025 +0.002 20.4240
Val 0.114 0.068 0.232 0.193 +0.015 20.1125
Trp 0.043 0.027 0.059 0.074 +0.007 20.4244
Phe 0.048 0.029 0.088 0.059 +0.005 20.3989
Iso 0.047 0.028 0.104 0.061 +0.006 20.3812
Leu 0.093 0.062 0.225 0.112 +0.010 20.2521
Lys 0.209 0.122 0.312 0.213 +0.024 0.4760
Total 3.543 2.237 6.271 2.492 +0.196 20.4000
Total essential amino acids 1.188 0.771 2.416 1.650 +0.131 20.2899
Total non-essential amino
acids
2.330 1.296 4.150 0.843 +0.074 20.5460
Amino acid content of human uterine fluid 919
at University of Southampton on January 9, 2016http://humrep.oxfordjournals.org/Downloaded from
n¼5). The fertile controls were matched as closely as possible to the
subfertile group; their average age was 29 years and BMI was 27.5 kg/m
2
.
Statistical analysis
Statistical analysis was performed using SPSS Statistics 21 (IBM, USA). The
data were shown to be not normally distributed using the Anderson –
Darling normality test. Results were expressed as an amino acid concentra-
tion median +interquartile range (IQR). Statistical analysis was performed
using either Mann –Whitney U-test or Kruskal–Wallis test as appropriate;
to determine whether the amino acid composition was affected by stage of
cycle, age, BMI, prudent diet score or gynaecological history. P≤0.05 was
considered significant. Spearman’s rank correlation was used to examine
the relationship between the concentrations of amino acids in the serum
and those in the uterine fluid in paired samples.
Results
Concentration of amino acids in human
uterine fluid
The concentrations of 18 amino acids in 56 human uterine fluid samples
were determined using reverse phase HPLC (Table I). The mean age of
women participating in the study was 32 years (range 19–45 years); the
mean BMI was 25.7 kg/m
2
(range 17.9 – 43.6 kg/m
2
). Glutamate was
found to be present in the highest concentration followed by glycine
and alanine. In contrast, methionine and tryptophan were found to be
present in the lowest concentration. In total, human uterine fluid was
observed to contain an amino acid concentration of 3.54 mM (IQR:
2.27–6.24 mM).
There was no statistically significant difference between the amino acid
concentrations found in the uterine fluid of fertile versus subfertile
women (P¼0.807). Amino acid profiles were also compared
between women of proven fertility or who had successfully conceived
following assisted reproductive techniques (n¼23) and those who did
not (n¼33). No significant difference was seen between these two
groups (P¼0.511).
Female diet alters the concentration
of amino acids in the uterine fluid
The short diet questionnaire was analysed and participants were given a
score based on their answers. In total, 21 females were categorized as
having an overall healthy diet while 25 were shown to have an unhealthy
Figure 1 Diet impacts the amino acid composition of uterine fluid in women. The effect of a negative or positive prudent diet score on the (A) individual
amino acids, (B) sum of total amino acids, (C) essential and (D) non-essential amino acids in the uterine fluid. Values are median +interquartile range.
n¼25 for a negative and n¼21 for a positive diet score. *P,0.05.
920 Kermack et al.
at University of Southampton on January 9, 2016http://humrep.oxfordjournals.org/Downloaded from
diet. The remaining 10 women had missing data meaning it was not pos-
sible to calculate a prudent diet score, and these weretherefore removed
from analysis. There was a significant difference in the uterine fluid con-
centration of eight amino acids between women with a positive prudent
diet score (healthy diet) when compared with those with a negative one
(unhealthy diet); asparagine (P¼0.018); histidine (P¼0.011); serine
(P¼0.033); glutamine (P¼0.049); valine (P¼0.025); phenylalanine
(P¼0.019); isoleucine (P¼0.025); and leucine (P¼0.043). Significantly
higher concentrations of these amino acids were seen in those with an
unhealthy diet compared with those who have a healthier diet (Fig. 1).
The results also demonstrated a significantly higher concentration of
branched chain amino acids in the uterine fluid of women with a negative,
compared with a positive, prudent diet score (P¼0.030; Fig. 2) but
there was no difference in the concentration of either essential or non-
essential amino acids between diet types.
Effect of menstrual cycle stage on the amino
acid content of human uterine fluid
There was no significant difference in the concentration of amino acids in
human uterine fluid between the proliferative stage (n¼35) and the se-
cretory stage (n¼18) of the menstrual cycle (Fig. 3A).
Effect of BMI on the amino acid content
of human uterine fluid
The concentration of amino acids in uterine fluid was measured in
women with a BMI of ,20 kg/m
2
(n¼6), women with a BMI within
the normal range (20 –25 kg/m
2
n¼29) and those with a BMI over
25 kg/m
2
(n¼21). Although there was a trend towards increased con-
centrations of valine, isoleucine and leucine with a rise in BMI, there were
no statistically significant differences between the groups (Fig. 3B). This
remained true when the groups were corrected for pathology or no
pathology, and fertile or subfertile.
Effect of gynaecological pathology on uterine
fluid amino acid content
Study participants were divided into those with no known pathology
(n¼24) (and either normal fertility or unexplained subfertility) and
those with pathology diagnosed either on ultrasound scan or during an
operation (n¼32). Pathology included submucosal uterine leiomyomas
(n¼3), ovarian pathology including simple cysts (n¼4) or PCOS (n¼
16), endometriosis (n¼3) and hydrosalpinx (n¼6). No statistically sig-
nificant difference in the amino acid concentration in uterine fluid was
demonstrated between the groups.
Effect of female age on the concentration
of amino acids in human uterine fluid
The total concentration of amino acids in human uterine fluid in women
under 38 years of age (n¼47) was not different to that in women older
than 38 years (n¼9), when a decline in fertility is thought to occur. This
remained true when the presence or absence of pathologies were taken
into account and when the subfertile group were compared with fertile
controls.
A comparison of the amino acid composition
of human serum and uterine fluid
No significant difference was seen between the amino acid concentration
of the serum in women who fasted prior to venepuncture and those who
did not. The concentration of each amino acid measured was similar in
the uterine fluid to that in serum, except for glutamate and aspartic
acid which showed more than a 20-fold increase in the uterine fluid
when compared with the serum. There was no correlation between
the concentration of individual amino acids measured in paired serum
and the uterine fluid samples (Table I).
Discussion
This study reports for the first time the amino acid content of human
uterine fluid. It is shown that this is stable through the menstrual cycle,
and changes little with increasing reproductive age, BMI or in the pres-
ence of a number of benign pathologies. In contrast however, diet is
shown to alter amino acid concentration in the uterine fluid and hence,
presumably, the nutritional composition within the reproductive tract
during preimplantation embryo development.
The techniques used in this study have the advantage that the uterine
fluid was collected directly, without the use of lavage. This, together with
amino acid measurement using sensitive reverse phase HPLC detection,
therefore increases the accuracy of the results obtained. However, a dis-
advantage of not using uterine flushing was the increased chance of
obtaining insufficient sample for analysis.
Studies using a number of animal models suggest that amino acid con-
centrations in the reproductive tract are actively regulated. The concen-
tration of amino acids found in murine (Harris et al., 2005), ovine (Gao
et al., 2009) and bovine (Fahning et al., 1967;Shorgan, 2003;Hugentobler
et al., 2007) uterine fluid has been described; however, no one has yet
characterized the amino acid profile in human uterine fluid using this
methodology. Although the concentrations of amino acids found in
human uterine fluid were similar to those observed in the mouse, differ-
ences in the levels of aspartate, glutamine, arginine, glycine, alanine were
Figure 2 Diet affects the levels of branched chain amino acids in
human uterine fluid. Graph demonstrating the difference in concentra-
tions of branched chain amino acids between women with a negative or
positive prudent diet score. Values are median +range. n¼25 for a
negative and n¼21 for a positive diet score. *P,0.05.
Amino acid content of human uterine fluid 921
at University of Southampton on January 9, 2016http://humrep.oxfordjournals.org/Downloaded from
observed, highlighting a variation between species (Harris et al., 2005). In
murine studies, amino acid concentration has been found to vary de-
pending on the site within the reproductive tract; higher levels observed
in the oviduct when compared with the uterine cavity (Harris et al.,
2005). Further studies demonstrated a difference in amino acid concen-
trations throughout the menstrual cycle in sheep between Days 3 and 16
of the cycle (Gao et al., 2009), and an increase in essential amino acids in
pregnancy in bovine research (Groebner et al., 2011). These variations in
amino acid concentration with cycle stage and pregnancy suggest that
levels in large animal models are regulated, which is in contrast to our
observations in the human where no differences were obtained
between the proliferative and secretory stages. However, previous
work in the human has shown a difference in taurine levels between
the mid-cycle and the luteal phase (Casslen, 1987).
Animal studies have also compared the concentration of amino acids
found in the Fallopian tube and uterine fluid. In both mice (Harris et al.,
2005) and cows (Elhassan et al., 2001), higher concentrations of amino
acids were found in the Fallopian tubes. In contrast, the human uterine
Figure 3 Stage of menstrual cycle and female BMI do not affect the amino acid composition of uterine fluid. Effect of (A) menstrual cycle stage and (B)
female BMI on the amino acid composition of human uterine fluid. Values are median +interquartile range. (A) For stage of cycle; n¼35 for proliferative
stage. n¼18 for secretory stage. (B) For BMI; n¼6 for a BMI ,20 kg/m
2
.n¼29 for a BMI between 20 and 25 kg/m
2
.n¼21 for a BMI over 25 kg/m
2
.
922 Kermack et al.
at University of Southampton on January 9, 2016http://humrep.oxfordjournals.org/Downloaded from
fluid amino acid concentrations presented in the current work were
higher for all amino acids than those reported in the fluid of the human
Fallopian tube (Tay et al., 1997). This finding is likely to reflect differences
in the techniques used to collect the fluid. In the current study, fluid was
obtained in situ using an embryo transfer catheter during a HyCoSy inves-
tigation or in theatre whereas Tay et al. (1997) collected Fallopian tubal
fluid following perfusion, which thus may account for their reduced levels.
There was no correlation between the concentrations of amino acids
observed in uterine fluid and the serum which suggests that the uterus
is a protected environment, and is in agreement with work in the
mouse (Eckert et al., 2012). In both the human and the mouse, glutamate
and aspartic acid demonstrated a .20-fold increase in the uterine fluid
when compared with the serum.
Although several studies report a decreased clinical pregnancy rate
with increased BMI (van der Steeg et al., 2008) and age (Leridon,
2004), a direct effect of diet and lifestyle on the amino acid concentration
of uterine fluid has not previously been investigated. Our results suggest
that the uterus is a protected environment and the amino acid concen-
tration is not altered by age, BMI or pathology.
Our data show that the amino acid composition of uterine fluid is sig-
nificantly influenced by a woman’s diet; a positive prudent diet score
(healthier diet) was associated with a significant reduction in asparagine,
histidine, serine, glutamine, valine, phenylalanine, isoleucine and leucine.
These findings are comparable to what has previously been observed in
mouse models fed a low protein diet, where reductions in the branched
chain amino acids were also observed (Eckert et al., 2012). This suggests
that, like the mouse, the nutritional environment of human uterine fluid is
sensitive to female diet.
This study has not investigated whether a higher or lower amino acid
concentration in human uterine fluid improves conception rates, or
whether it is the homeostasis of the environment that is essential.
However, the quiet embryo hypothesis suggests that a low amino acid
turnover improves embryo development (Houghton et al., 2002;
Leese, 2002). Recently it has been shown that the decidualised endomet-
rium acts as a biosensor of embryo quality (Brosens et al., 2014). It could
therefore be proposed that women with a more prudent diet may
promote a low amino acid environment in utero which selectively sup-
ports the development of high quality, metabolically quiet embryos.
This is of particular relevance as in this study, serine and leucine
showed a statistically significant reduction in the uterine fluid of
women with a positive prudent diet score when compared with those
with a negative score, and these amino acids have previously been
demonstrated as predictors of embryo viability (Brison et al., 2004).
These data offer the potential to facilitate the production of embryo
culture media containing physiologically relevant concentrations of
amino acids based on those found in uterine fluid, and perhaps also to
guide preconception dietary interventions to optimize the intrauterine
environment. In clinical practice, IVF laboratories use embryo culture
media whereby the nutritional content has been extrapolated from
data obtained from murine embryo development (Gardner and Lane,
1998) and thus may not reflect the nutritive requirement of the dev-
eloping human embryo as it moves through the reproductive tract
(Houghton, 2012). Given that there is now significant, albeit con-
troversial (Carrasco et al., 2013;Lin et al., 2013), evidence supporting
the profound influence of the preimplantation environment on subse-
quent birthweight (Dumoulin et al., 2010;Eskild et al., 2013), it is pos-
sible that the inclusion of physiological concentrations of pleiotropic
nutrients, such as amino acids, could further enhance the success of
clinical IVF.
In conclusion, these data provide the first evidence to suggest that dif-
ferences in women’s diet quality can alter the amino acid concentration
of human uterine fluid. Further research is required to examine the
impact of the human periconception diet on both the uterine environ-
ment and embryo development.
Acknowledgements
We are grateful to Dr Sian Robinson and Dr Sarah Crozier for their help
with the use and analysis of the Southampton Women’s survey diet ques-
tionnaire and Kate Parry for technical support. A.J.K. was supported by
the University of Southampton National Institute of Health Research
Academic Clinical Fellowship Scheme.
Authors’ roles
Y.C.C., J.J.E., N.S.M. and F.D.H. conceived the experiments. A.J.K.,
Y.C.C., N.S.M., J.J.E. and F.D.H. designed the experiments. A.J.K. and
S.F.-S. performed the experiments. N.B. and Y.C.C. collected samples.
A.J.K. and S.F.-S. analysed the data. All authors were involvedin the prep-
aration of the manuscript.
Funding
This work was funded by the NIHR, the Medical Research Council
(G0701153), Infertility Research Trust and the University of Southamp-
ton. This report is independent research by the National Institute for
Health Research Biomedical Research Centre Funding Scheme. The
views expressed in this publication are those of the author(s) and not
necessarily those of the NHS, the National Institute for Health Research
or the Department of Health. Funding to pay the Open Access publica-
tion charges for this article was provided by the MRC UK.
Conflict of interest
None declared.
References
Alexiou M, Leese HJ. Purine utilisation, de novo synthesis and degradation in
mouse preimplantation embryos. Development 1992;114:185–192.
Barker DJ. Developmental origins of adult health and disease. J Epidemiol
Community Health 2004;58:114–115.
Barker DJ. The origins of the developmental origins theory. J Intern Med 2007;
261:412–417.
Boomsma CM, Kavelaars A, Eijkemans MJ, Amarouchi K, Teklenburg G,
Gutknecht D, Fauser BJ, Heijnen CJ, Macklon NS. Cytokine profiling in
endometrial secretions: a non-invasive window on endometrial
receptivity. Reprod Biomed Online 2009;18:85– 94.
Brison DR, Houghton FD, Falconer D, Roberts SA, Hawkhead J,
Humpherson PG, Lieberman BA, Leese HJ. Identification of viable
embryos in IVF by non-invasive measurement of amino acid turnover.
Hum Reprod 2004;19:2319–2324.
Brosens JJ, Salker MS, Teklenburg G, Nautiyal J, Salter S, Lucas ES, Steel JH,
Christian M, Chan YW, Boomsma CM et al. Uterine selection of human
embryos at implantation. Sci Rep 2014;4:3894.
Amino acid content of human uterine fluid 923
at University of Southampton on January 9, 2016http://humrep.oxfordjournals.org/Downloaded from
CarrascoB, Boada M, RodriguezI, Coroleu B, Barri PN, Veiga A. Doesculture
medium influence offspring birth weight? Fertil Steril 2013;100:1283– 1288.
Casslen BG. Free amino acids in human uterine fluid. Possible role of high
taurine concentration. J Reprod Med 1987;32:181– 184.
Chen X, He J, Ding Y, Zeng L, Gao R, Cheng S, Liu X, Wang Y. The role of
MTOR in mouse uterus during embryo implantation. Reproduction 2009;
138:351–356.
Christensen DR, Calder PC, Houghton FD. Effect of oxygen tension on the
amino Acid utilisation of human embryonic stem cells. Cell Physiol
Biochem 2014;33:237–246.
Crozier SR, Inskip HM, Barker ME, Lawrence WT, Cooper C, RobinsonSM.
Development of a 20-item food frequency questionnaire to assess a
‘prudent’ dietary pattern among young women in Southampton. Eur
J Clin Nutr 2010;64:99–104.
Dawson KM, Collins JL, Baltz JM. Osmolarity-dependent glycine
accumulation indicates a role for glycine as an organic osmolyte in early
preimplantation mouse embryos. Biol Reprod 1998;59:225–232.
Devreker F, Hardy K, Van den Bergh M, Vannin AS, Emiliani S, Englert Y.
Amino acids promote human blastocyst development in vitro.Hum
Reprod 2001;16:749– 756.
Dumoulin JC, Land JA, Van Montfoort AP, Nelissen EC, Coonen E,
Derhaag JG, Schreurs IL, Dunselman GA, Kester AD, Geraedts JP et al.
Effect of in vitro culture of human embryos on birthweight of newborns.
Hum Reprod 2010;25:605–612.
Eckert JJ, Porter R, Watkins AJ, Burt E, Brooks S, Leese HJ, Humpherson PG,
Cameron IT, Fleming TP. Metabolic induction and early responses of
mouse blastocyst developmental programming following maternal low
protein diet affecting life-long health. PLoS One 2012;7:e52791.
Edwards LJ, Williams DA, Gardner DK. Intracellular pH of the mouse
preimplantation embryo: amino acids act as buffers of intracellular pH.
Hum Reprod 1998;13:3441–3448.
Elhassan YM, Wu G, Leanez AC, Tasca RJ, WatsonAJ, Westhusin ME. Amino
acid concentrations in fluids from the bovine oviduct and uterus and in
KSOM-based culture media. Theriogenology 2001;55:1907 –1918.
Eskild A, Monkerud L, Tanbo T. Birthweight and placental weight; do changes
in culture media used for IVF matter? Comparisons with spontaneous
pregnancies in the corresponding time periods. Hum Reprod 2013;
28:3207–3214.
Fahning ML, Schultz RH, Graham EF. The free amino acid content of uterine
fluids and blood serum in the cow. J Reprod Fertil 1967;13:229– 236.
GaoH,WuG,SpencerTE,JohnsonGA,LiX,BazerFW.Selectnutrientsin
the ovine uterine lumen. I. Amino acids, glucose, and ions in uterine
lumenal flushings of cyclic and pregnant ewes. Biol Reprod 2009;80:
86–93.
Gardner DK, Lane M. Culture of viable human blastocysts in defined
sequential serum-free media. Hum Reprod 1998;13(Suppl 3):148 –159;
discussion 160.
Groebner AE, Rubio-Aliaga I, Schulke K, Reichenbach HD, Daniel H, Wolf E,
Meyer HH, Ulbrich SE. Increase of essential amino acids in the bovine uterine
lumen during preimplantation development. Reproduction 2011;141:685– 695.
Harris SE, Gopichandran N, Picton HM, Leese HJ, Orsi NM. Nutrient
concentrations in murine follicular fluid and the female reproductive
tract. Theriogenology 2005;64:992– 1006.
Houghton FD. Media composition: amino acids and cellular homeostasis.
Methods Mol Biol 2012;912:97–106.
Houghton F. Identification of viable embryos by noninvasive measurement of
amino acids in culture media. In: Gardner DK, Sakkas D, Seli E, Wells D
(eds). Human Gametes and Preimplantation Embryos. New York: Springer,
2013, 267–273.
Houghton FD, Hawkhead JA, Humpherson PG, Hogg JE, Balen AH,
Rutherford AJ, Leese HJ. Non-invasive amino acid turnover predicts
human embryo developmental capacity. Hum Reprod 2002;17:999– 1005.
Hugentobler SA, Diskin MG, Leese HJ, Humpherson PG, Watson T,
Sreenan JM, Morris DG. Amino acids in oviduct and uterine fluid and
blood plasma during the estrous cycle in the bovine. Mol Reprod Dev
2007;74:445–454.
Kwong WY, Wild AE, Roberts P, Willis AC, Fleming TP. Maternal
undernutrition during the preimplantation period of rat development
causes blastocyst abnormalities and programming of postnatal
hypertension. Development 2000;127:4195–4202.
Lane M, Gardner DK. Differential regulation of mouse embryo development
and viability by amino acids. J Reprod Fertil 1997;109:153 – 164.
Lane M, Gardner DK. Amino acids and vitamins prevent culture-induced
metabolic perturbations and associated loss of viability of mouse
blastocysts. Hum Reprod 1998;13:991– 997.
Leese HJ. Quiet please, do not disturb: a hypothesis of embryo metabolism
and viability. Bioessays 2002;24:845–849.
Leridon H. Can assisted reproduction technology compensate for the natural
decline in fertility with age? A model assessment. Hum Reprod 2004;
19:1548–1553.
Lin S, Li M, Lian Y, Chen L, Liu P. No effect of embryo culture media on
birthweight and length of newborns. Hum Reprod 2013;28:1762–1767.
Manser RC, Leese HJ, Houghton FD. Effect of inhibiting nitric oxide
production on mouse preimplantation embryo development and
metabolism. Biol Reprod 2004;71:528–533.
Nasr-Esfahani MH, Winston NJ, Johnson MH. Effects of glucose, glutamine,
ethylenediaminetetraacetic acid and oxygen tension on the concentration of
reactive oxygen species and on development of the mouse preimplantation
embryo in vitro.J Reprod Fertil 1992;96:219– 231.
Shorgan LRWLWSB. Free amino acid content of oviductal and uterine fluid at
different oestrous stages in the cow. Curr Zool 2003;49:80–85.
Stokes PJ, Hawkhead JA, Fawthrop RK, Picton HM, Sharma V, Leese HJ,
Houghton FD. Metabolism of human embryos following cryopreservation:
implications for the safety and selection of embryos for transfer in clinical
IVF. Hum Reprod 2007;22:829– 835.
Sun C, Velazquez MA, Marfy-Smith S, Sheth B, Cox A, Johnston DA, Smyth N,
Fleming TP. Mouse early extra-embryonic lineages activate compensatory
endocytosis in response to poor maternal nutrition. Development 2014;
141:1140–1150.
Tay JI, Rutherford AJ, Killick SR, Maguiness SD, Partridge RJ, Leese HJ. Human
tubal fluid: production, nutrient composition and response to adrenergic
agents. Hum Reprod 1997;12:2451–2456.
van der Steeg JW, Steures P, Eijkemans MJC, Habbema JDF, Hompes PGA,
Burggraaff JM, Oosterhuis GJE, Bossuyt PMM, van der Veen F, Mol BWJ.
Obesity affects spontaneous pregnancy chances in subfertile, ovulatory
women. Hum Reprod 2008;23:324– 328.
Watkins AJ, Ursell E, Panton R, Papenbrock T, Hollis L, Cunningham C,
Wilkins A, Perry VH, Sheth B, Kwong WY et al. Adaptive responses by
mouse early embryos to maternal diet protect fetal growth but
predispose to adult onset disease. Biol Reprod 2008;78:299 – 306.
924 Kermack et al.
at University of Southampton on January 9, 2016http://humrep.oxfordjournals.org/Downloaded from
