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Int. J. Environ. Res. Public Health 2020, 17, 2445; doi:10.3390/ijerph17072445 www.mdpi.com/journal/ijerph
Article
Consumption of a Branched-Chain Amino Acid
(BCAA) during Days 2–10 of Pregnancy Causes
Abnormal Fetal and Placental Growth: Implications
for BCAA Supplementation in Humans
Chiu Yuen To
1,2
, Muriel Freeman
2,3
and Lon J. Van Winkle
2,4,
*
1
Department of Surgery, Division of Neurosurgery, St Francis Hospital, Memphis, TN 38119, USA;
tochiuyuen@gmail.com
2
Department of Biochemistry, Midwestern University, Downers Grove, IL 60515, USA;
muriel.freeman@my.rfums.org
3
Department of Surgery, Division of Podiatric Medicine and Surgery, Carle Richland Memorial Hospital,
Olney, IL 62450, USA
4
Department of Medical Humanities, Rocky Vista University 8401 S. Chambers Road, Parker, CO 80134, USA
* Correspondence: lvanwinkle@rvu.edu
Received: 21 March 2020; Accepted: 30 March 2020; Published: 3 April 2020
Abstract: A relatively large branched-chain amino acid (BCAA) supplement, consumed for more
than 10 days, appears to be especially effective at alleviating muscle damage and soreness during
intense human training. However, perturbations in amino acid and protein consumption could have
unwanted transgenerational effects on male and female reproduction. This paper hypothesizes that
isoleucine consumption by female mice from days 2 to 10 of pregnancy will alter fetal and placental
growth later in gestation. Mice that had received 118 mM isoleucine in their drinking water
delivered pups on day 19 of pregnancy that were 9% larger than normal, whereas the reverse was
true for pups born on day 20. Moreover, the inverse correlation between birth weight and litter size
was lost in mice that previously consumed excess isoleucine. Similarly, the normal correlations
between fetal and placental weights were lost by day 18 of pregnancy in mice that had consumed
excess isoleucine. Mice that consumed excess isoleucine had placentas smaller than, and fetuses
larger than normal on day 18 of pregnancy, but the reverse was true on day 15. Other unintended
and unexpected effects of BCAA consumption should be studied more thoroughly due to the
increasing use of BCAAs to alleviate muscle damage and soreness in athletes.
Keywords: branched-chain amino acids; isoleucine; fetus; placenta; small-for-gestational-age;
embryo; muscle; exercise training
1. Introduction
Dietary branched-chain amino acid (BCAA) supplementation is becoming a solution to several
human health problems [1–8]. Excess consumption of BCAAs reduces muscle soreness from exercise [1–3],
counters fatigue during exercise [4], and alleviates exercise-induced skeletal muscle damage [1,3,5].
Higher BCAA levels are also associated with a lower prevalence of obesity [6,7], and their increased
consumption improves liver function in patients undergoing liver surgery [8].
Alterations in protein and amino acid intake can, however, adversely influence embryo
development with transgenerational consequences in adulthood [9–11]. For example, the
consumption of a low protein diet by pregnant rats led to the development of larger than normal
fetuses on day 19 of pregnancy, but their growth was not sustained, and they were smaller than the
Int. J. Environ. Res. Public Health 2020, 17, 2445 2 of 10
control fetuses by day 21 of pregnancy [12]. Both small and large-for-gestational-age offspring of
mammals, including humans, are predisposed to develop metabolic syndrome and related disorders
in adulthood [10,11,13]. Hence, we studied whether the altered consumption of a BCAA during
pregnancy can influence the growth and development of mouse embryos.
A relatively large BCAA supplement, consumed for more than 10 days, appears to be especially
effective at alleviating muscle damage and soreness during intense human training [3]. In some
studies, male and female athletes more than doubled their intake of BCAAs [1,2]. Other studies
included an additional amino acid, such as arginine, in their supplements (e.g., [14]). For these
reasons, we simplified our approach by limiting this study to a single BCAA, and we more than
doubled the isoleucine (Ile) intake by mice between days 2 and 10 of pregnancy. Ile was selected, in part,
because it is less biologically active than leucine, which serves as a signaling molecule via mammalian
target of rapamycin (mTOR) [15]. While some amino acids are known to regulate embryo development
through one-carbon and other aspects of metabolism in stem cells [11], nothing is known about how
Ile might influence these and other cells.
We performed two studies: In study 1, we determined whether the consumption of Ile from days
2 to 10 of pregnancy alters the birth weights of mouse pups. Study 2 was designed to determine
whether Ile consumption leads to abnormal fetal and placental growth. We hypothesized that the
consumption of Ile by female mice from days 2 to 10 of pregnancy will change the fetal and placental
growth later in gestation, resulting in small- or large-for-gestational-age offspring.
2. Methods
Sexually mature Swiss ICR female mice (Harlan Sprague-Dawley, Inc., Indianapolis, IN, USA)
were allowed to acclimate to a 14 h light–10 h dark cycle for at least 2 weeks [16,17]. They were then
placed with a fertile male, and natural ovulation and mating were confirmed by the presence of a
copulatory plug the following morning (day one of pregnancy). Two groups of mice were included
in study 1; one group of 12 experimental (E) mice received 118 mM Ile in their drinking water from
days 2 to 10 of pregnancy, while another 12 served as control (C) mice and drank regular water. All
mice consumed Purina rodent chow ad libitum. Pregnancies were otherwise allowed to proceed
normally, and the pups delivered on days 19 and 20 were weighed immediately.
Subsequent experiments in study 2 involved four groups of 8 to 12 mice each. The pups of two
groups were delivered via caesarian sections on day 15 of pregnancy, with one group (the
experimental mice) having drunk 118 mM Ile-treated water from days 2 to 10 of pregnancy, and the
other (the control mice) having consumed regular water. Similarly, the conceptuses from the other
groups of control (C) and experimental (E) mice were obtained on day 18. Daily water and food intake
were measured in the groups of mice that underwent caesarian sections on day 18. At the time of
delivery, the conceptuses were carefully dissected in an attempt to preserve the integrity of the
amniotic membranes for the measurement of the weights of the whole conceptuses. The weights of
the whole conceptuses, placentas, and fetuses were measured upon delivery. Figure 1 displays a
summary of our scheme to collect the fetal and placental weights on day 18 of pregnancy. A similar
approach was used to collect data on day 15.
Data were analyzed statistically using contingency tables, t-tests, determination of Pearson
correlation coefficients (r values), and analyses of variance (ANOVA) combined with multiple
comparison tests as appropriate (GraphPad Prism 8.0.2 Software, Inc., La Jolla, CA, USA). Effect sizes
were also calculated as r values.
Data were analyzed on both per conceptus/offspring and per dam bases, as there is controversy
regarding whether the unit of dietary treatment of pregnant mice, or each of their
conceptuses/offspring, is the dam [18–20]. In the case of per dam analysis, the mean weights of the
fetuses, placentas, and offspring from a given dam were calculated, and these means were used in
statistical analyses as single pieces of data. Consequently, sample sizes in the latter cases equal the
number of dams, rather than the number of conceptuses/offspring. When the sample size is made
larger by comparing the means for individual conceptuses/offspring, rather than the number of dams,
Int. J. Environ. Res. Public Health 2020, 17, 2445 3 of 10
the level of statistical significance is, of course, higher. Data were reported as means + 95% confidence
intervals (CI).
Means of fetal
and placental
weights
(n = 164 each)
Means per
litter and then
mean of litters
(n = 12 litters)
Fetal/placental
weight ratios
Paired
fetal and
placetal
weights
164 con ceptuses
Twelve experime ntal
mice consumed 118mM
L-isole ucine during
days 2-10 of pregnancy
Means per
litter and then
mean of litters
(n = 9 litters)
Fetal/placental
weight ratios
Paired
fetal and
placental
weights
Means of fetal
and placental
weights
(n = 128 each)
128 con ceptuses
Nine con trol
mice co nsumed
regular water during
days 2-10 of pregnancy
21 Pre gnant Mice
Figure 1. The experimental (E) and control (C) mice that underwent caesarian sections on day 18 of
pregnancy.
These studies were approved by the Midwestern University Institutional Animal Care and Use
Committee (MWU File Numbers 1486 and 1560).
3. Results
The experimental mice gained 28.73 + 3.49 g between days 1 and 18 of pregnancy, while the
control mice gained 28.59 + 4.64 g (see food and water consumption below). In study 1, four control
mice delivered pups on day 19 of pregnancy, and eight delivered pups on day 20. Conversely, eight
experimental mice delivered pups on day 19 of pregnancy, while four delivered pups on day 20. The
pups of the experimental (E) mice were about 9% heavier than those pups born to the control (C) mice
on day 19 (E19 vs. C19 in Table 1). On day 20, however, pups born to the experimental mice were 9%
lighter than pups of the control mice (E20 vs. C20 in Table 1). Thus, pups of the experimental mice were
large-for-gestational-age on day 19, but they were small-for-gestational-age when born on day 20.
Furthermore, birth weights were inversely correlated with litter size in the control mice (r = −0.65, p < 0.05)
but not in the experimental mice (r = −0.05, ns).
Table 1. Mean ± 95% confidence interval birth weights for the control (C) and experimental (E)
offspring born on day 19 (C19 and E19) and 20 (C20 and E20).
Per Offspring (g/pup) Per Dam (g/pup/dam)
C19 1.34 ± 0.03
a
n
= 60 1.35 ± 0.10
a
n
= 4
C20 1.66 ± 0.03
b
n
=102 1.66 ± 0.06
b
n
= 8
E19 1.47 ± 0.03
c
n
=106 1.47 ± 0.08
c
n
= 8
E20
1.51 ± 0.05
c
n = 58
1.52 ± 0.10
c
n = 4
a,b,c
Superscripts indicate mean weights that are significantly different (analyses of variance
(ANOVA) with multiple comparison tests, p < 0.0001 per offspring, p < 0.05 per dam).
In study 2, the fetal weights were positively correlated with the placental weights in both the
control (r = 0.43, p < 0.001) and experimental (r = 0.48, p < 0.001) mice on day 15, but this correlation was
Int. J. Environ. Res. Public Health 2020, 17, 2445 4 of 10
lost in the experimental mice (but not the control mice) by day 18 (r = 0.06, ns in the experimental mice
vs. r = 0.34, p < 0.001 in the control mice). The change in the r values for the experimental mice between
days 15 and 18 was also statistically significant (Fisher r-to-z transformation, p < 0.001). The sizes of
placentas increased in the control mice between days 15 and 18 of pregnancy, but such was not the
case for the experimental mice (Figure 2). Moreover, fetuses were larger in the control mice than in
the experimental mice on day 15, but the reverse was true on day 18 (Figure 3). Similarly, fetal/placental
weight ratios per dam were lower in the experimental mice than in the control mice on day 15, but the
opposite was true on day 18 (Figure 4).
The experimental mice also had an increased fragility of fetal membranes on day 18 of pregnancy,
as indicated by the percentage of membrane ruptures during dissection. Dissections were performed
carefully in an attempt to preserve the integrity of the whole conceptuses for weighing, and the
investigators had no preconceived notion that such an event would occur more frequently in one
group than another. When performing caesarian sections, there was a 60% greater incidence of unwanted
rupturing of amniotic membranes in the experimental mice than in the control mice on day 18 (Figure 5).
Figure 2. Placentas grew significantly between days 15 and 18 of pregnancy in the control mice (t-test,
p < 0.0001), but not in the experimental mice. Double asterisks (**) indicate mean values that are
significantly different from each other (control day 15, n = 95 placentas and day 18, n = 128 placentas;
experimental day 15, n = 123 placentas and day 18, n = 164 placentas). CI—confidence interval.
(a) (b)
Int. J. Environ. Res. Public Health 2020, 17, 2445 5 of 10
900
1000
1100
1200
1300
1400
1500
1600
1700
0
20
40
60
Distribution of Experimental (E) Fetal Weights
on Day 18 of Pregnancy
Bin Center (fetal weight, mg)
Number of values
(c) (d)
Figure 3. The experimental (E) fetuses were significantly smaller than the normal, control (C) fetuses
on day 15 of pregnancy (a; t-test, p < 0.0001), but they were larger than normal on day 18 (b; t-test, p <
0.0001). Double asterisks (**) indicate mean values that are significantly different from each other.
Also shown are the distributions of weights on day 18 for the control (C, c) and experimental (E, d)
fetuses. (See Figure 2 for sample sizes).
Figure 4. The fetal/placental weight ratio on a per dam basis was larger in the control than in the
experimental (isoleucine) mice on day 15 (t-test, p < 0.001), but this ratio was larger in the experimental
than in the control mice on day 18 (t-test, p < 0.0001). Double asterisks (**) indicate mean values that
are significantly different from each other (control day 15, n = 8 dams and day 18, n = 9 dams;
isoleucine day 15, n = 10 dams and day 18, n = 12 dams).
Int. J. Environ. Res. Public Health 2020, 17, 2445 6 of 10
Figure 5. Conceptuses ruptured more frequently than normal (control) during the dissection of the
experimental (isoleucine) mice on day 18 of pregnancy (contingency table, p < 0.01; mean ± 95% CI
ruptured conceptuses per dam in control vs. isoleucine mice, t-test after arcsine transformation of the
data, p < 0.05). A single asterisk (*) indicates mean values that are significantly different from each
other. (See Figures 2 and 4 for sample sizes).
Ile supplementation between days 2 and 10 of pregnancy did not greatly alter water and food
intake by the mice between days 2 and 18 of gestation, as shown in Figures 6 and 7. Nor did
differences in water and food intake seem to account for differences in the fetal and placental weights
in the control and experimental mice on day 15 of pregnancy. The increase in food intake by the
experimental mice on day 17 as displayed in Figure 7 may, however, have supported more rapid than
normal fetal growth between days 15 and 18 of pregnancy, shown in Figure 3.
Figure 6. Daily water intake of the control (C) and experimental (E) mice that delivered on day 18 of
pregnancy. The difference between the two groups is statistically significant only on day 3 (t-test, p < 0.001).
Double asterisks (**) indicate mean values that are significantly different from each other (control
mice, n = 10 dams; experimental mice, n = 10 dams). The number of mice is higher in Figure 4 for
experimental mice because water intake was measured reliably in only 10 mice, and the number is lower
for control mice in Figure 4 because one mouse delivered on day 18, before dissection could occur.
Int. J. Environ. Res. Public Health 2020, 17, 2445 7 of 10
Figure 7. Daily food intake of the control (C) and experimental (E) mice that delivered on day 18 of
pregnancy. The difference between the two groups is statistically significant on day 3 (t-test, p < 0.05).
In addition, a significant increase in food intake occurred in the experimental mice on day 17 (ANOVA
with multiple comparison tests for all E data including 16E vs. 17E, p < 0.0001), but such was not the
case in the control mice until day 18. Single and double asterisks (*, **) indicate mean values that are
significantly different from each other (control mice, n = 10 dams; experimental mice, n = 10 dams).
The number of mice is higher in Figure 4 for experimental mice because food intake was measured
reliably in only 10 mice, and the number is lower for control mice in Figure 4 because one mouse
delivered on day 18, before dissection could occur.
4. Discussion
Ile consumption by female mice for 10 days after mating caused multiple changes later in
pregnancy and even after gestation. These changes included increases and decreases in the weights
of the resultant fetuses, placentas, and offspring, shown in Table 1 and Figures 2–4. For pup weights,
the effect sizes for ANOVA on a per dam or per offspring basis were r = 0.77 and 0.58, respectively,
and are of crucial practical importance [21]. For the direct comparison of the control and experimental
offspring born on day 20, these numerically adjacent mean values had effect size values on a per dam and
per offspring basis of r = 0.63 and 0.50, respectively, and are also of crucial practical importance [21].
Extraembryonic membranes also appeared to become more fragile as a result of prior Ile supplementation,
shown in Figure 5. Hence, the unintended effects of BCAA supplementation to support strength training
in humans may occur after the period during which more BCAAs are consumed.
The current results using a mouse model may seem, at first, to apply more to reproductive-age
females than male athletes. However, perturbations in amino acid and protein consumption by males
also adversely affect the offspring they sire [11,22]. Moreover, the full effects of BCAA
supplementation on athletes remain to be established. Although positive effects on training have been
observed, studies are needed to determine whether BCAAs have immediate, as well as longer-term,
detrimental impacts.
Such effects seem especially likely to occur in processes involving stem cells. Perturbations in stem
cell function occur as a result of challenges to protein and amino acid metabolism and signaling [9–11].
The present results show that Ile supplementation may produce such challenges, as evidenced by the
abnormal growth of mouse fetuses due to prior Ile consumption by their mothers.
However, by what mechanism might Ile supplementation given to mice during the first half of
pregnancy alter fetal and placental growth closer to the conclusion of gestation? One possibility is
the partial Ile inhibition of leucine-stimulated mTOR signaling during the preimplantation blastocyst
development period [15]. Subsequently altered peri-implantation development, due to this challenge
to amino acid metabolism and signaling, could lead to abnormal placental function and fetal growth
later on, as is the case for low protein diets [9–11,15]. Such abnormal placental function likely includes
inhibition of placental insulin, mTOR, and signal transducer and activator of transcription (STAT)
signaling, and the resultant down-regulation of amino acid transporter expression [23,24]. The effects
Int. J. Environ. Res. Public Health 2020, 17, 2445 8 of 10
of excess Ile consumption are likely to be more complex; however, the experimental mouse fetuses in
our study exhibited both slower and more rapid growth than the normal mouse fetuses depending on
the period of development, as shown in Figure 3.
Moreover, the effects of protein and amino acid challenges are not always intuitively obvious or
easy to predict. For example, maternal consumption of a low protein diet does not alter the
concentration of BCAAs in rat fetuses, but the addition of threonine to the low protein diet
significantly lowers the concentrations of these amino acids in the fetuses [25]. The spectra of possible
effects of protein and amino acid perturbations, such as from BCAA supplementation, warrant
further exploration, especially since their effects may be transgenerational [10,11].
5. Limitations
We studied the effects of a single BCAA on growth and development in mice. Thus, it is a
challenge to extrapolate our findings to other species, or to other BCAAs and mixtures of them.
Nevertheless, the developmental origins of health and disease (Barker) hypothesis applies well to all
mammalian species including humans [9–11]. According to this well-documented theory, maternal
and paternal lifestyle changes, such as an increase or decrease in dietary amino acid consumption,
regulate early embryo development through both genetic and epigenetic modifications. These
modifications can last a lifetime, and can be passed to future generations. Moreover, environmental
challenges act through epigenetic changes in stem cells in both human and rodent embryos [9–11].
Hence, it seems prudent to study the effects of dietary BCAAs more broadly, in both rodent models
as well as humans who consume BCAAs to improve their own health. Even the beneficial changes
associated with BCAA consumption may result, in part, from altered stem cell function in adults.
6. Conclusions
We verified our hypothesis that Ile consumption by female mice from day 2 to 10 of pregnancy
alters fetal and placental growth later in gestation. Ile supplementation led to slower than normal
growth of fetuses up to day 15 of pregnancy, but then faster growth between days 15 and 18 of
gestation. Conversely, Ile consumption produced large-for-gestational-age offspring on day 19 of
pregnancy, but pups born on day 20 were smaller than normal. Abnormal placental development
likely contributed to this atypical fetal growth pattern. We suggest that Ile supplementation, around
the time of embryo implantation on day 5 of gestation, started aberrant placentation by altering
leucine-signaling via mTOR.
Author Contributions: Conceptualization, L.J.V.W.; Data curation, C.Y.T. and M.F.; Formal analysis, C.Y.T.,
M.F. and L.J.V.W.; Methodology, C.Y.T., M.F. and L.J.V.W.; Supervision, L.J.V.W.; Writing – original draft,
C.Y.T., M.F. and L.J.V.W.; Writing—review and editing, C.Y.T., M.F. and L.J.V.W. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no potential conflicts of interest with respect to the research,
authorship, and/or publication of this article.
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