Maturitas 69 (2011) 91–93

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Maturitas
journal homepage: www.elsevier.com/locate/maturitas

Birth-weight as a risk factor for cancer in adulthood: The stem cell perspective
C. Capittini a,∗ , P. Bergamaschi a , A. De Silvestri b , A. Marchesi a , V. Genovese a , B. Romano a ,
C. Tinelli b , L. Salvaneschi a
a
Pavia Cord Blood Bank, Transfusion Medicine Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
b
Statistics Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The ‘stem cell burden’ hypothesis represents a plausible explanation for the association between birth-
Received 8 October 2010 weight and the risk of breast cancer in adulthood. The size of the overall stem cell pool would be expected
Received in revised form 1 February 2011 to affect organ size and consequently birth-weight, making birth-weight a proxy for the overall number
Accepted 8 February 2011
of fetal stem cells. As stem cells are self-renewing, the greater their number is at birth, the higher will be
the chance that one of them will undergo carcinogenesis over the years. To investigate the correlation
between birth-weight and stem cell burden, we examined the cord blood hematopoietic CD34+ stem
Keywords:
cell population as an indicator of the overall fetal stem cell number. We measured both the CD34+ level
Cancer risk
Birth weight
(by flow cytometry) and the CD34+ proliferative potential (by the GM-CFU culture), in a sample of 1037
Stem cell healthy newborn cord blood donors. We found that heavier babies had a significantly greater CD34+
Proliferative potential stem cell concentration (p < 0.001) and a higher GM-CFU number than lighter babies (p < 0.001). Thus, a
high birth-weight was positively associated with a high concentration of CD34+ stem cells and also with
a qualitatively higher “stemness” of this pool. Therefore, our data support the theory that birth-weight
reflects the number of fetal stem cells.
© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction To investigate the correlation between birth-weight and stem

It has been argued that early life factors, such as birth-weight
and rapid growth during infancy, can affect the lifetime risk of
breast cancer [1]. An intriguing link between birth-weight and
the risk of breast cancer in adulthood is represented by the ‘stem
cell burden’ hypothesis, first proposed by Trichopoulos and Lip-
worth [2]. Somatic stem cells are present in the breast (and indeed
many other tissues) from fetal development, and as they are long-
living and self-renewing they are especially likely, over a lifespan,
to undergo oncogenic mutation. The higher the number of breast
stem cells at birth, the greater will be the possibility that one of
these cells will mutate [3]. Furthermore, it has been reported that a
general increase in the stem cell pool can affect organ size, and con-
sequently birth-weight [4]. Thus, although birth-weight itself does
not play a causal role in the development of cancer, it may be an
indicator of stem cell number, and thereby be positively associated
with adult cancer risk (not only of breast cancer but essentially for
all tissues containing stem cell niches).
∗ Corresponding author. Tel.: +39 0382 503088.
E-mail address: cristina.capittini@libero.it (C. Capittini).
cell burden, we examined the cord blood hematopoietic CD34+
stem cell population as an indicator of the overall fetal stem cell
number.
2. Materials and methods
2.1. Study subjects
Our study examined a total of 1037 healthy newborns regis-
tered at the Cord Blood Bank of Pavia (IRCCS Foundation Policlinico
San Matteo, Pavia, Italy). All mothers gave their signed voluntary
informed consent to participate in the cord blood banking pro-
gram for unrelated stem cell transplantation. Following the Bank’s
protocols, a specifically trained physician obtained a complete
medical history of the newborn’s family (mother, father, siblings
and grandparents). An exhaustive obstetric history (i.e. of previous
pregnancies) was also obtained, so that we could exclude women
with recurrent spontaneous abortions from donation. Furthermore,
during each pregnancy the health of both the mother and the
fetus was carefully monitored, in order to exclude from donation
any stem cells derived from pathological pregnancies, including
preterm ones (<37 gestational weeks).

0378-5122/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.maturitas.2011.02.013
92 C. Capittini et al. / Maturitas 69 (2011) 91–93
2.2. Cord blood collection
Immediately after birth, cord blood was collected from umbil-
ical vessels by in utero technique; only samples of 50 ml or more
and containing at least 800 × 106 nucleated cells were banked for
transplantation purposes.
2.3. Analysis of hematopoietic stem cell populations by flow
cytometry
We measured the population of hematopoietic progenitor stem
cells by flow cytometry, detecting the expression of the CD34 and
CD45 antigens. Cells were stained with CD34 (PE) and CD45 (FITC)
antibodies (BD Biosciences, San José, CA, USA), and the CD34+
cell count was determined using both single- and double-platform
methods, according to the guidelines of the International Society of
Hematotherapy and Graft Engineering (ISHAGE) [5].
2.4. Analysis of hematopoietic stem cell potential by GM-CFU
assay
Hematopoietic progenitors are able to form colonies in vitro
when stimulated by specific growth factors. The proliferative
potential of CD34+ stem cells is expressed by the growth of colonies
derived from granulocyte monocyte-colony forming units (GM-
CFUs). Thus, the GM-CFU assay is a complementary analysis which
can usefully confirm the results of flow cytometry, and it also
identifies CD34+ clonogenicity. The procedure consists of setting
up duplicate short-term cell cultures in a semi-solid medium
(MethoCult® GF H84444, StemCell Technologies, Vancouver, BC,
Canada). Cord blood cells are added to the culture medium to reach
a plating concentration of 1.7 × 104 cells per 35 mm dish. For 14
days, the plates are incubated at 37 ◦ C in an atmosphere of 5% CO2
and >95% humidity. The colonies are then counted and classified.
2.5. Percentiles based on birth-weight, gestational age and gender
Birth-weight, gender and gestational weeks of the 1037 new-
borns were obtained from hospital records. Following the fetal
growth curves in use at the Obstetric-Gynecological Clinic of the
IRCCS Foundation of the Policlinico San Matteo of Pavia [6], each
baby was assigned to a percentile, according to birth-weight, ges-
tational age and gender. Then, the 1037 infants were divided into
four percentile groups: <25, 25–49.99, 50–75, >75.
2.6. Statistical analysis
First, we studied birth-weight as a categorical variable. We
estimated the geometric means for the CD34+ stem cell concen-
tration and the GM-CFU count within four percentile groups: <25,
25–49.99, 50–75, >75. We calculated the percentage difference in
these parameters between the first and the fourth group, taking
into consideration a 95% confidence interval (CI). Subsequently, we
treated birth-weight as a continuous variable, and considered how
a 25-point percentile increase in birth-weight would affect the stem
cell count. We then used a parametric regression to examine the
association between birth-weight (using the <25 percentile group
as the reference) and CD34+ stem cell concentration and GM-CFU
count. Statistical significance was set at 0.05.
3. Results
We analyzed the birth-weight and cord blood CD34+ stem cell
data of 1037 full-term infants registered in the Cord Blood Bank of
Pavia.
Table 1
The geometric means of CD34+ stem cell concentration (CD34+/␮L) and GM-CFU
×104 count are shown in the four birth-weight percentile groups: <25, 25–49.99,
50–75, >75. SD: standard deviation.
CD34+/␮L GM-CFU ×104
Mean SD Mean SD
<25 27.77 20.49 304.82 257.37
25–49.99 34.96 28.39 395.83 359.20
50–75 36.96 28.31 445.41 431.21
>75 37.58 28.48 501.17 519.57
Table 1 shows the geometric means of the CD34+ stem cell
concentration and GM-CFU count in each of the four percentile
groups. The first group (<25 percentile) had a mean CD34+ stem cell
count of 27.77 per ␮L (standard deviation, SD, 20.49), and a mean
GM-CFU count of 304.82 × 104 (SD 257.37). The fourth group (>75
percentile) had a mean CD34+ stem cell count of 37.58 per ␮L (SD
28.48) and a mean GM-CFU count of 501.17 × 104 (SD 519.57). The
percentage difference in these parameters between the first and
the fourth percentile group was 35% (95% CI 24–46) for the CD34+
stem cell concentration and 67% (95% CI 47–84) for the GM-CFU
count.
We then treated birth-weight as a continuous variable and con-
sidered estimates for a 25-point percentile increase in birth-weight.
Using a parametric regression, we examined the association
between CD34+ stem cell concentration and GM-CFU count across
the whole range of birth-weight percentiles. We found a posi-
tive association between birth-weight percentile groups and both
CD34+ stem cell concentration (p < 0.001) and GM-CFU count
(p < 0.001). Each 25–point percentile increase in birth-weight was
associated with a 16.0% (95% CI 7.2–34.8%) higher CD34+ stem
cell concentration and a 61.0% (95% CI 49.5–73.5%) higher GM-CFU
count.
4. Discussion
Birth-weight is positively associated with cancer risk, in partic-
ular breast cancer risk, in adulthood [1]; one explanation for this
association is that birth-weight is an indicator of the size of the
pool of long-living and self-renewing fetal stem cells, which are
candidates for oncogenic mutations over the lifespan [2–4].
We analyzed the correlation between birth-weight and both the
CD34+ stem cell concentration and the clonogenic potential (GM-
CFU count) of this stem cell pool for a sample of 1037 cord blood
infant donors. Our babies were born from physiological pregnancies
and were all healthy at birth.
We first considered the mean values of CD34+ stem cell concen-
tration and GM-CFU count in four percentile groups: <25, 25–49.99,
50–75, >75. Going from the first to the fourth group, we found a
gradual increase in the mean parameters: heavier babies had both
a greater CD34+ stem cell concentration and a higher GM-CFU count
than lighter babies (Table 1).
The impact of CD34+ stem cell concentration and proliferation
on birth-weight appears evident, particularly for clonogenicity, as
the percentage differences in these parameters between the first
and the fourth percentile groups were 35% and 67%, respectively.
In order to identify a good marker for the overall number of
fetal stem cells, Strohsnitter and colleagues measured hematopoi-
etic CD34+ stem cell levels in the cord blood of 288 term newborns
[7]. The authors found a positive association between birth-weight
and CD34+ stem cell concentration: each 500 g increase in weight
at birth was associated with a 15.5% increase in the number of stem
cells in cord blood samples.
In our parametric regression analysis we found a positive asso-
ciation not only between CD34+ stem cell concentration and
percentile groups (p < 0.001), but also between GM-CFU count and
 C. Capittini et al. / Maturitas 69 (2011) 91–93
percentile groups (p < 0.001). Further, each 25-point percentile
increase in birth-weight was associated with a 16% higher stem
cell concentration and with a 61% higher clonogenic potential in
cord blood samples.
These data support previous findings of a direct correlation
between birth-weight and hematopoietic CD34+ stem cell num-
ber [7], and suggest that birth-weight correlates not only with fetal
stem cell density, but also with stem cell proliferation. That is, a
higher birth-weight is correlated not only with a quantitatively
higher concentration of CD34+ stem cells, but also with a quali-
tatively higher ‘stemness’ of this pool.
Our data provide further support for the theory that high birth-
weight reflects the fetal stem cell number, in accordance with the
stem cell burden hypothesis. Nevertheless, as the molecular and
cellular mechanisms underlying fetal stem cell pool expansion are
still poorly understood, many questions remain. What are the key
regulators of the fetal stem cell potential? Are these factors con-
nected in a single network, or do they belong to different pathways?
Is there a genetic or an epigenetic program influencing fetal stem
cell potential?
4.1. Closing remarks
As the intrauterine environment strongly influences the fetal
stem cell number, which in turn increases the adult cancer risk, it
seems logical to presume that the months spent by the fetus in the
uterus can influence future cancer risk.
Competing of interests
The authors declare that they do not have any conflict of inter-
ests.
Contributors
Cristina Capittini and Paola Bergamaschi wrote the manuscript;
Annalisa De Silvestri and Carmine Tinelli made statistical analyses;
93
Andrea Marchesi, Valeria Genovese, Bina Romano made CD34+
stem cell and GM-CFU assays; Laura Salvaneschi is the Direc-
tor of the Pavia Cord Blood Bank and revised both data and
manuscript.
Funding
The authors Cristina Capittini, Paola Bergamaschi, Annal-
isa De Silvestri, Andrea Marchesi, Valeria Genovese, Bina
Romano, Carmine Tinelli, and Laura Salvaneschi declare no
funding.
Acknowledgements
We wish to thank the two anonymous reviewers for their con-
structive comments on the draft of this manuscript, and Dr. Anna
Bertoni and Dr. Francesca Cattaneo, our proof-readers. A special
thank for the contribution of Mrs. Linda Johns for the English
revision.
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