The American Journal of Medicine (2005) 118, 899-906

CLINICAL RESEARCH STUDY

Male microchimerism in women without sons:

Quantitative assessment and correlation with pregnancy
history
Zhen Yan, MD, PhD,a Nathalie C. Lambert, PhD,a,f Katherine A. Guthrie, PhD,b
Allison J. Porter, BA,a Laurence S. Loubiere, PhD,a Margaret M. Madeleine, PhD,c
Anne M. Stevens, MD, PhD,a,d Heidi M. Hermes, BS,a,d and J. Lee Nelson, MDa,e

a
Program in Human Immunogenetics, bClinical Statistics, and cProgram in Cancer Epidemiology Research Cooperative, Fred
Hutchinson Cancer Research Center, Seattle, Wash. dDepartment of Pediatrics and eDepartment of Medicine, Rheumatology,
University of Washington, Seattle, Wash. fLaboratoire d’ Immuno-rhumatologie, INSERM U639, Marseille, France.

KEYWORDS: ABSTRACT
Microchimerism; PURPOSE: Fetal microchimerism, derived from fetal cells that persist after pregnancy, is usually
Pregnancy; evaluated by tests for male microchimerism in women who gave birth to sons. We investigated male
Y chromosome microchimerism in women without sons and examined correlation with prior pregnancy history.
Immunologic consequences of microchimerism are unknown. We studied healthy women and women
with rheumatoid arthritis (RA).
METHODS: Y-chromosome–specific real-time quantitative polymerase chain reaction was used to
test peripheral blood mononuclear cells of 120 women (49 healthy and 71 with RA). Results were
expressed as the number of male cells that would be equivalent to the total amount of male DNA
detected within a sample containing the equivalent of 100 000 female cells.
RESULTS: Male microchimerism was found in 21% of women overall. Healthy women and women
with RA did not significantly differ (24% vs 18%). Results ranged from the DNA equivalent of 0 to 20.7
male cells per 100 000 female cells. Women were categorized into 4 groups according to pregnancy
history. Group A had only daughters (n ⫽ 26), Group B had spontaneous abortions (n ⫽ 23), Group
C had induced abortions (n ⫽ 23), and Group D were nulligravid (n ⫽ 48). Male microchimerism
prevalence was significantly greater in Group C than other groups (8%, 22%, 57%, 10%, respectively).
Levels were also significantly higher in the induced abortion group.
CONCLUSIONS: Male microchimerism was not infrequent in women without sons. Besides known
pregnancies, other possible sources of male microchimerism include unrecognized spontaneous abor-
tion, vanished male twin, an older brother transferred by the maternal circulation, or sexual intercourse.
Male microchimerism was significantly more frequent and levels were higher in women with induced
abortion than in women with other pregnancy histories. Further studies are needed to determine specific
origins of male microchimerism in women.
© 2005 Elsevier Inc. All rights reserved.

Supported by National Institutes of Health grants AI41721, AR39282, Immunogenetics D2-100, Fred Hutchinson Cancer Research Center, 1100
and AI45659. Fairview Ave N, P.O. Box 19024, Seattle, WA 98109.
Requests for reprints should be addressed to Zhen Yan, MD, PhD, E-mail address: zyan@fhcrc.org.

0002-9343/$ -see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.amjmed.2005.03.037
900
Some fetal cells reach the maternal circulation during preg-
nancy and persist decades later, creating fetal microchimer-
ism.1-3 Microchimerism refers to the state of an individual
who harbors 2 genetically distinct and separately derived
cell populations, 1 of which is present at a low concentra-
tion. Cells also traffic from mother to fetus, and maternal
microchimerism has been described in the mother’s progeny
in adult life.1,2,4-6 Another potential source of naturally
acquired microchimerism from pregnancy is from a twin.7
Iatrogenic microchimerism occurs as a consequence of or-
gan transplantation (eg, kidney, liver, and heart),8 and per-
sistent donor microchimerism has been reported after blood
transfusion.9
Investigations of persistent fetal microchimerism have
primarily been conducted examining parous women. How-
ever, women experience a range of different outcomes from
pregnancy, and little is known about fetal microchimerism
after pregnancies that do not continue to term. Moreover,
fetal microchimerism is usually measured by testing for
male DNA or male cells in women who previously gave
birth to a son because of the technical issue that 1 test can
be used to study many women.10 However, male microchi-
merism in women without sons is not well defined, nor is
any potential difference in microchimerism, depending on
the type of pregnancy outcome.
The primary purpose of the current studies was to deter-
mine the prevalence and levels of male microchimerism in
women with no prior birth of a son and to evaluate results
for correlation with prior pregnancy history. Immunologic
consequences of naturally acquired microchimerism from
pregnancy are as yet unknown and potentially may include
both adverse and beneficial effects. Fetal microchimerism
has been adversely implicated in scleroderma, an autoim-
mune disease with clinical similarity to graft-versus-host
disease after hematopoietic cell transplantation.11 Microchi-
merism has not been investigated in rheumatoid arthritis
(RA), but studies have reported a reduction in RA risk in
association with pregnancy, suggesting the possibility of a
beneficial effect.12 We tested healthy women and women
with RA for male microchimerism in peripheral blood
mononuclear cells (PBMCs) with the use of real-time
quantitative polymerase chain reaction (Q-PCR) for a
Y-chromosome–specific sequence, DYS14. Male micro-
chimerism was examined for correlation with prior preg-
nancy history of spontaneous abortion, induced abortion,
birth of daughters only, and among nulligravid women.
The prevalence (any positive result) and quantitative lev-
els of male microchimerism were evaluated.
Patients and methods
Study participants
A total of 120 women were studied, including 49 healthy
women and 71 women with RA. For each study subject a
The American Journal of Medicine, Vol 118, No 8, August 2005
trained interviewer completed a 1.5-hour in-person inter-
view with comprehensive detailing of all aspects of repro-
ductive history, including confirmation by the creation of a
calendar that specified each reproductive event for duration
and chronology. The criteria for acceptance to the study was
completion of this interview and that a woman had no
history of having given birth to a son. Subjects with RA met
the American College of Rheumatology criteria for RA and
were identified as part of a previous prospective, popula-
tion-based study of reproductive factors in newly diagnosed
RA in women.13,14 Healthy women were recruited as part of
other studies from a similar geographic distribution (Seattle,
Wash, and surrounding area) using similar methods.15 In
addition, 5 female children who had no pregnancies and no
history of sexual intercourse, aged between 7 and 15 years
old, and blood from 2 umbilical cords of female newborns
were also studied. Ethics approval was acquired from the
institutional review board, and informed consent was ob-
tained from participating volunteers.
The majority of study subjects were white. Among
healthy women, 46 of 49 were white; among women with
RA, 64 of 71 were white. Others were African American
(one healthy woman, one with RA), Asian (one healthy
woman, four with RA), Native American (two with RA),
and mixed ethnicity (one healthy woman). Women were
categorized according to pregnancy history into 4 groups:
Groups A, B, C, and D (Table 1). Among RA patients with
pregnancies, the majority occurred before RA disease onset
(32/38). No woman with RA had a male twin; information
regarding a twin was unavailable for healthy women. No
study subject had an organ transplantation.
Procurement of blood specimens and preparation
of peripheral blood mononuclear cells
Peripheral venous blood samples were drawn into acid citrate
dextrose solution A-vacutainer tubes. PBMCs were isolated
from the whole blood by dilution in Hank’s balanced salt
solution and Ficoll Hypaque (Pharmacia Biotech, Uppsula,
Sweden) gradient centrifugation at a density of 1.077 g/mL.
DNA extraction from peripheral blood
mononuclear cells
Genomic DNA was extracted from PBMCs under a biosafety
hood using a Wizard Genomic DNA Purification Kit (Pro-
mega, Madison, Wis), in accordance with the manufacturer’s
instructions, and resuspended in 10 mM Tris-HCL buffer
(tris[hydroxymethyl]aminomethane-hydrochloride, pH 9.0).
Detection of male DNA by real-time quantitative
polymerase chain reaction
The DYS14 sequence was selected as a Y-chromosome–
specific marker to detect male microchimerism. The meth-
ods for the real-time Q-PCR have been described by Lam-
 Yan et al
Male microchimerism in women without sons
Table 1 Characteristics of women who never gave birth to a son at the time of blood draw, by group, healthy women (HW) and women with rheumatoid arthritis (RA)*
Gestational age of last
Age at Age at abortion (week)‡ Drugs used at time of study‡
last last Time since Time since Age at
Subjects gravidity abortion No. of No. of No. of last gravidity last abortion RA onset None or Immune
Group no. Age (yrs)¶ (yrs)¶ (yrs)¶ pregnancies† abortions† live births† 1-12 13-24 Uk (yrs)† (yrs)† (yrs)¶ NSAID affecting Uk

Healthy women
HW-A 9 43 ⫾ 12 27 ⫾ 5 NA 1 (1-2) 0 1 (1-2) NA NA NA 12 (1-39) NA NA 9 (100) 0 (0) 0 (0)
HW-B 13 51 ⫾ 11 28 ⫾ 8 27 ⫾ 8 2 (1-4) 1 (1-4) 0 (0–2) 9 (69) 2 (15) 2 (15) 21 (3-53) 24 (3-53) NA 13 (100) 0 (0) 0 (0)
HW-C 12 42 ⫾ 10 27 ⫾ 9 27 ⫾ 9 1 (1-3) 1 (1-3) 0 11 (92) 0 (0) 1 (8) 12 (3-42) 12 (3-42) NA 12 (100) 0 (0) 0 (0)
HW-D 15 31 ⫾ 8 NA NA 0 0 0 NA NA NA NA NA NA 15 (100) 0 (0) 0 (0)
Women with RA
RA-A 17 44 ⫾ 11 28 ⫾ 5 NA 2 (1-3) 0 2 (1-3) NA NA NA 14 (0-44) NA 39 ⫾ 9 3 (18) 13 (76) 1 (6)
RA-B 10 48 ⫾ 14 30 ⫾ 4 25 ⫾ 4 3 (1-8) 1 (1-7) 1 (0-5) 6 (60) 2 (20) 2 (20) 16 (1-36) 27 (1-42) 41 ⫾ 12 6 (60) 4 (40) 0 (0)
RA-C 11 38 ⫾ 11 28 ⫾ 7 26 ⫾ 8 2 (1-5) 1 (1-3) 0 (0-3) 10 (91) 1 (9) 0 (0) 11 (1-21) 11 (3-28) 34 ⫾ 9 3 (27) 6 (55) 2 (18)
RA-D 33 43 ⫾ 10 NA NA 0 0 0 NA NA NA NA NA 36 ⫾ 10 14 (42) 19 (58) 0 (0)

*Group A: HW or RA who had only given birth to daughters and had no history of spontaneous or induced abortion; Group B: HW or RA with a history of clinically recognized spontaneous abortion and
no history of induced abortion; Group C: HW or RA with a history of induced abortion and no history of spontaneous abortion; Group D: nulligravid HW or RA women. The term abortion describes both induced
and spontaneous events. NSAID: non-steroidal anti-inflammatory drug.
¶Mean ⫾ standard deviation.
†Median (range).
‡Number (percentage).
NA ⫽ not applicable.
Uk ⫽ Unknown.

901
902
bert et al.16 The sensitivity of our assay is detection of
approximately 1 male cell in 100 000 female cells. Speci-
ficity of the DYS14 primers was validated by testing the
primers with commercially purified male or female genomic
DNA (Promega) and confirmed that only male DNA was
amplified in Q-PCR.
Six aliquots of DNA were tested with DYS14 primers for
each study subject. To quantitatively measure male DNA, the
Y-chromosome–specific calibration curve was run on each
plate, using a dilution of the equivalent DNA of 500, 100, 50,
10, 5, 1, or 0.5 male cells in a background of 20 000 female
cells. Two additional DNA aliquots were amplified concur-
rently for ␤-globin to define the total DNA concentration of the
sample tested. The standard ␤-globin curve was run with a
dilution of the equivalent DNA of 50 000, 25 000, 10 000,
5000, 1000, 500, or 100 cells. An acceptable range of the total
cells tested per aliquot was considered to be 5000 to 30 000
(larger amounts of DNA can result in inhibition). The conver-
sion factor used was 6.6 pg of DNA per cell. Standard curves
for ␤-globin and DYS14 were run simultaneously. The amount
of male microchimerism was expressed as the number of male
cells that would be equivalent to the total amount of male DNA
detected within a sample containing the equivalent of 100 000
female cells (male genomic equivalent cells per 100 000 fe-
male cells abbreviated McEq/100 000). Because the male mi-
crochimerism in each aliquot is assumed to have a Poisson
distribution, the average number of male cells per 100 000
female cells for each subject was calculated by combining
information across aliquots of DNA as previously described.16
The mean of total number of cells tested was similar across
groups. Among healthy women, the mean was 116 083 cells in
Group A, 116 578 in Group B, 100 675 in Group C, and
126 086 in Group D. Among women with RA, the mean in
Group A was 88 820 cells (somewhat lower), 99 034 in Group
B, 101 986 in Group C, and 118 237 in Group D.
Q-PCR reactions were set up in a reaction volume of 50
␮L. Each reaction contained 5 ␮L DNA, 5 ␮L 10⫻platinum
buffer (Applied Biosystems, Foster City, Calif), 300 nM of
each amplification primer, 100 nM dual-labeled probe, 200
nM of each deoxynucleoside triphosphate, 3.5 mM MgCl2,
1.5 U AmpliTaq Gold DNA polymerase (Applied Biosys-
tems), and 60 nM Rox reference dye (Synthegen, Houston,
Tex). The amplification conditions consisted of an initial
incubation at 50°C for 2 minutes, followed by 95°C for 10
minutes, and 45 cycles of 95°C for 15 seconds and 60°C for
1 minute. An Applied Biosystems 7000 sequence detector
collected the amplification data, which were analyzed by
means of Sequence Detection System software (Applied
Biosystems).
Sequencing of polymerase chain reaction products
PCR products from some positive women were cloned into
the pCR2.1-TOPO vector using the TOPO TA Cloning Kit
(Invitrogen, Carlsbad, Calif) and sequenced. Sequencing
reactions were performed on an ABI 377 Automated DNA
Sequencer (Applied Biosystems).
The American Journal of Medicine, Vol 118, No 8, August 2005
Anti-contamination procedures
Rigorous precautions against PCR contamination were
taken. Aerosol-resistant pipette tips and clean gloves were
always used. Isolation of DNA and setup of amplification
reactions were accomplished in separate locations by female
technicians who were blinded to the clinical context of
samples with no knowledge of pregnancy history. The pos-
sibility of carryover contamination was minimized by use of
the 7000 sequence detector, because its optical detection
system precludes the need to reopen the reaction tubes after
amplification. It was required that at least 2 different ali-
quots from a sample demonstrate results above threshold
level to be considered positive. Multiple negative controls
were included in each Q-PCR plate with no DNA and with
DNA from a nulligravid female (known negative). Negative
controls were consistently negative across all of the exper-
imental plates. A positive control of male genomic DNA
was also included in each experiment.
Statistical analysis
Two methods of analysis were used for investigation of
differences in microchimerism across groups of healthy
women or women with RA. First, a binary outcome of
whether male microchimerism was detected was analyzed
with exact logistic regression models. For the second anal-
ysis, microchimerism was used as a continuous measure in
linear regression. To account for departures from normality
in the distribution of the microchimerism values, the ranks
of the values were used as the outcome in linear regression
models. The following factors were considered as potential
confounders: age at draw date, gravidity (ⱖ1 vs 0), age at
last gravidity, gestational week of last abortion (ⱖ13 vs
1-12, n ⫽ 22 healthy women, n ⫽ 19 RA), time from last
gravidity to draw date, and interval from last abortion to
draw date. Gestational age and/or date of abortion was
unknown for a few women. For women with RA, age at
disease onset and use of medications at time of testing
(immune affecting, eg, hydroxychloroquine, methotrexate,
cyclophosphamide, and prednisone, vs not immune affect-
ing) were also considered as potential confounders.
Results
Y-chromosome–specific real-time quantitative PCR was
used to test DNA extracted from PBMCs of 120 women, 49
who were healthy and 71 who had RA, all of whom had no
history of prior birth of a son. Male microchimerism was
found in 21% of women overall. The prevalence of male
microchimerism did not significantly differ in healthy
women and women with RA, 24% and 18%, respectively.
No male DNA was found in female children and umbilical
cord blood from female newborns, which were tested as
additional controls. Study women were classified into 4
Yan et al Male microchimerism in women without sons 903

Figure 1 Levels of male microchimerism in peripheral blood mononuclear cells from healthy women (HW) (A) and women with
rheumatoid arthritis (RA) (B) who had never given birth to a son. Group A: healthy women or women with RA who had only given birth
to daughters; Group B: healthy women or women with RA with a history of spontaneous abortion; Group C: healthy women or women with
RA with a history of induced abortion; Group D: nulligravid healthy women or women with RA.

groups according to prior reproductive histories as de-
scribed in Methods. Table 1 shows the characteristics of
each cohort. With respect to duration of gestation, the ma-
jority of the women who had spontaneous or induced abor-
tion were in their first trimester (gestational age 1-12
weeks). Among healthy women, Group C subjects were
somewhat younger than Group B subjects, and Group D
subjects were younger on average than other healthy women
group subjects. Except for those factors related to group
assignments, we found no statistically significant differ-
ences in any of the variables indicated in Table 1 across
groups of women with RA.
Sequencing of the PCR-amplified products of positive
women demonstrated 100% homology to human Y-chro-
mosome DYS14 sequence. Among the 12 healthy women
who were positive for male microchimerism, 3 were from
Group B, 7 were from Group C, and 2 were from Group D
(Figure 1, A). In pairwise comparisons among healthy
women (Table 2), the probability of male microchimerism
was significantly higher in Group C (ie, with a history of
induced abortion) (58%) than in Group A (0%) (P ⫽ .01) or
Group D (13%) (P ⫽ .04). Although the comparison be-
tween Group C and B was not significant (P ⫽ .16), the
contrast was similar to those of other groups. Thus, healthy
904 The American Journal of Medicine, Vol 118, No 8, August 2005

Table 2 Prevalence of male DNA in women who never gave

Results were next examined for quantitative differences
birth to a son, by group, healthy women (HW) and women (ie, differences in levels of male microchimerism). Among
with rheumatoid arthritis (RA) healthy women, levels of male microchimerism were all
zero in Group A, from 0 to 0.1 McEq/100 000 (median 0) in
Subjects No. (%) Group B, 0 to 1.6 McEq/100 000 (median 0.1) in Group C,
Group no. positive P value* and 0 to 0.5 McEq/100 000 (median 0) in Group D (Figure
Healthy women n ⫽ 49 1, A). No significant quantitative difference was observed
HW-A 9 0 (0) 0.01 across Groups A, B, and D. In pairwise comparisons (Table
HW-B 13 3 (23) 0.16 3), male microchimerism levels were significantly higher in
HW-C 12 7 (58) –
HW-D 15 2 (13) 0.04 Group C than in Group A (P ⫽ .003), Group B (P ⫽ .04),
Women with RA n ⫽ 71 or Group D (P ⫽ .01). Thus, subjects from Group C (n ⫽
RA-A 17 2 (12) 0.02 12) were compared with all other healthy women (Groups
RA-B 10 2 (20) 0.1 A, B, and D, n ⫽ 37). In an unadjusted analysis, induced
RA-C 11 6 (55) – abortion was significantly associated with higher male mi-
RA-D 33 3 (9) 0.004
crochimerism levels, compared with the other groups (P ⬍
*Among healthy women, the P value was calculated as compared to .001). This estimate was not materially changed by adjust-
Group HW-C.
Among women with RA, the P value was calculated as compared to
ment for any of the potential confounders described in
Group RA-C. Methods.
Results of analysis for male microchimerism levels were
similar among corresponding groups for women with RA.
women from Group C (n ⫽ 12) were compared with all Levels of male microchimerism ranged from 0 to 6.6 McEq/
other healthy women (Groups A, B, and D, n ⫽ 37). In an 100 000 in Group A (median 0), 0 to 5.6 McEq/100 000 in
unadjusted analysis, the odds of women in Group C having Group B (median 0), 0 to 20.7 McEq/100 000 in Group C
male microchimerism was estimated to be more than 8 (median 0.4), and 0 to 2.7 McEq/100 000 in Group D
times greater than the odds in other healthy women (odds (median 0) (Figure 1, B). No quantitatively significant dif-
ratio [OR] 8.4, 95% confidence interval [CI] 1.6-50.9, P ⫽ ference was found across Groups A, B, and D. In pairwise
.008). This estimate was not substantially altered by adjust- comparisons (Table 3), male microchimerism values were
ment for age at blood draw date or other factors as described significantly greater in the induced abortion group (Group
in Methods. C) than in Group A (P ⫽ .01) or Group D (P ⫽ .02).
The prevalence of male microchimerism was similar in Analysis suggested a similar trend toward higher levels in
corresponding categories of women with RA as in healthy
women. Of 13 women with RA who had male microchi-
merism, 2 were from Group A, 2 were from Group B, 6 Table 3 Levels of male DNA in women who never gave
were from Group C, and 3 were from Group D (Figure 1, birth to a son, by group, healthy women (HW) and women
B). Similar to healthy women, the frequency of any detect- with rheumatoid arthritis (RA)
able male microchimerism was significantly higher in
women with RA with induced abortions (Group C) (55%) DNA equivalent
number of male
than in Group A (12%) (P ⫽ .02) or Group D (9%) (P ⫽
cells per 100 000
.004). Although the comparison between Group C and female cells
Group B was not significant (P ⫽ .1), the contrast was (McEq/100 000)
similar to those of the other groups. Thus, the probability of Subjects
male microchimerism in women with RA in Group C (n ⫽ Group no. Median Range P value*
11) was compared with all other women with RA (Groups Healthy women
A, B, and D, n ⫽ 60). In an unadjusted analysis, women n ⫽ 49
with RA who had experienced induced abortion were 9.1 HW-A 9 0 0-0 0.003
times more likely to have male microchimerism than other HW-B 13 0 0-0.1 0.04
women with RA without a history of induced abortion (OR HW-C 12 0.1 0-1.6 –
9.1, 95% CI 2.2-37.8, P ⫽ .002). It is unknown whether HW-D 15 0 0-0.5 0.01
Women with RA
immunomodulatory medications affect the frequency or lev- n ⫽ 71
els of microchimerism. When we conducted an analysis RA-A 17 0 0-6.6 0.01
after adjustment for the use of immunomodulatory medica- RA-B 10 0 0-5.6 0.1
tions at the time of blood draw, the difference in women RA-C 11 0.4 0-20.7 –
with RA with induced abortion compared with other groups RA-D 33 0 0-2.7 0.02
remained statistically significant (adjusted OR 7.2, 95% CI Among women with RA, the P value was calculated as compared to
1.4-35.9, P ⫽ .02). Results were not substantially altered Group RA-C.
*Among healthy women, the P value was calculated as compared to
by adjustment for any of the other factors described in
Group HW-C.
Methods.
Yan et al Male microchimerism in women without sons
Group C compared with Group B (P ⫽ .1). Therefore, the
level of male microchimerism in women with RA from
Group C (n ⫽ 11) was further compared with all other
women with RA (Groups A, B, and D, n ⫽ 60). The results
indicated that women with RA who had a history of induced
abortion (Group C) were more likely to have higher levels of
male microchimerism, compared with women with RA in
other groups (P ⫽ .0004). Because our analysis used the ranks
of values, the high value in Group C (20.7 McEq/100 000) did
not unduly influence the result. Also, the result was not mate-
rially affected by adjustment for any of the potential confound-
ers described in Methods.
Discussion
Trafficking of cells and cell-free nucleic acids at the fetal-
maternal interface is now a well-documented phenomenon
during normal human pregnancy.1,2 Recent recognition of
persistent male microchimerism in the circulation of women
who gave birth to sons (presumed fetal microchimer-
ism),3,17 as well as in maternal tissues,18 is of interest
especially because long-term effects of microchimerism are
currently unknown. Studies of fetal microchimerism usually
use testing for male microchimerism in women who have
given birth to a son. This approach is used most often
because of the technical reason that a single assay can be
used to test many study subjects. In studies of microchimer-
ism in transplantation, similarly, the most common ap-
proach is to test for male microchimerism in women recip-
ients of grafts from males.10 However, the prevalence of
male microchimerism in women with pregnancy outcomes
other than a term pregnancy, as well as that among nulli-
gravid women, has not been well established. A review of
studies to date indicates male microchimerism has some-
times been identified in some women with no history of a
male birth.3,16,17,19 We therefore sought to determine the
prevalence of male microchimerism in women without a
male birth and to examine the prevalence and levels of male
microchimerism for correlation with prior pregnancy his-
tory in studies of healthy women and women with RA.
Overall, we found that slightly more than one fifth of all
women with no history of a male birth had male microchi-
merism in their peripheral blood. Among women who only
had daughters or were nulligravid, one potential explanation
for male microchimerism could be a nonrecognized (male)
miscarriage. Fetal traffic into maternal blood has been re-
ported as early as 4 to 5 weeks postconception,20 and recent
studies suggest the incidence of early pregnancy loss is
much higher than previously thought.21 A second potential
source is from a “vanished (male) twin.” A vanished twin is
thought to be a relatively common phenomena resulting
from spontaneous resorption of one sac or embryo in a twin
pregnancy.22 Twin loss occurs most often in the first tri-
mester and is usually completely reabsorbed into the pla-
centa without being noticed at birth.22 A third possibility is
905
from an older male sibling transferred by the maternal
circulation to the fetus of a later pregnancy. Another pos-
sibility that has not been investigated is whether male DNA
can be detected in a woman’s circulation from sexual inter-
course without pregnancy. In the current study, male mi-
crochimerism was not found in the peripheral blood of
female children with no history of pregnancy or sexual
activity or in the cord blood of female newborns. Some
positive results might be anticipated, however, should a
larger study specifically investigate this population because
of the potential for male microchimerism from a vanished
male twin or possibly from an older brother as described.
The primary purpose of our study was to investigate the
prevalence and levels of male microchimerism in women
without sons and to examine for correlation with pregnancy
history. Both the prevalence and levels of male microchi-
merism were greater in women with induced abortion com-
pared with women with other pregnancy histories. This
observation was significant among healthy women and
among women with RA. Neither qualitative nor quantitative
differences were observed across groups with other preg-
nancy histories including women who only had daughters,
women who had a spontaneous abortion or were nulli-
gravid, among healthy women or women with RA. En-
hanced transfer of fetus to mother traffic has been reported
in women undergoing elective pregnancy terminations.23 In
our study the majority of women with induced abortions
were in the first trimester (⬎90%). Both increased transfer
and a high proliferative capacity are potential explanations
for better engraftment and/or maintenance of fetal micro-
chimerism after an induced abortion; the latter possibility is
supported by the observation that proportionately more fetal
nucleated cells are CD34⫹ hematopoietic stem/progenitor
cells in early gestation than at term.24
The immunologic consequences of naturally acquired
microchimerism are not known, but both adverse and ben-
eficial consequences are subjects of ongoing investigation.
A potentially adverse role for fetal microchimerism has
been suggested in scleroderma.11,25,26 Microchimerism has
not been investigated in RA. In general, pregnancy is
thought to be beneficial to RA. A woman who has RA and
becomes pregnant will usually experience amelioration or
even disappearance of her arthritis during the pregnancy.27
In addition, a majority of studies have described a decreased
risk of RA among women who had children.12 However, in
the former instance RA recurs postpartum and in the latter
instance an increased incidence of new onset RA occurs in
the postpartum window.12 The effect of spontaneous or
induced abortion on RA is less clear. Whether microchi-
merism impacts the risk of RA will require larger and more
extensive studies in which the specific source of microchi-
merism is also identified.
Our study has a number of limitations. There are poten-
tial sources of male DNA that we were not able to evaluate
including from blood transfusion9 or from an older brother,
information that was unavailable to us. On the other hand it
906
is likely that our results underestimate the true prevalence of
microchimerism after induced or spontaneous abortion be-
cause the sex of lost fetuses is largely unknown. Another
issue that is always a concern when PCR-based techniques
are used is contamination. Although the possibility of con-
tamination cannot be entirely excluded, stringent measures
were used to minimize risk, all PCR tests were carried out
in a separate area and by female technicians, and negative
controls were consistently negative. The possibility of PCR
cross-reaction with an autosomal sequence was ruled out by
sequence analysis of positive PCR products.
In summary, male microchimerism was not uncommon
in the peripheral blood of healthy women and women with
RA who never gave birth to a son. Male microchimerism
was significantly more prevalent and present at higher levels
when the prior pregnancy was an induced abortion. Male
microchimerism was also sometimes found in women who
only had daughters, had miscarriages, or had no recognized
prior pregnancies. Our observations suggest caution is in-
dicated in attributing the source of male microchimerism in
studies of autoimmune disease or transplantation. Further
studies are needed to test for other genetic polymorphisms
to identify the specific origins of male microchimerism
sources and to explore any biologic effects of microchimer-
ism from pregnancy that persist decades later in women.
Acknowledgments
We are indebted to Jennifer Brackensick, Cathy Harper, and
Lisa Johnson for study coordination and to Meghan Mullar-
key, Jennifer Pang, and Timothy Erickson for technical
support.
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