Alterations in maternal-fetal cellular trafficking
after fetal surgery
Payam Saadaia, Tzong-Hae Leeb, Geoanna Bautistaa, Kelly D. Gonzalesa,
Amar Nijagala, Michael P. Buschb, Chong Jai Kimc, Roberto Romeroc,
Hanmin Leea, Shinjiro Hirosea, Larry Randa, Douglas Miniatia,
Diana L. Farmera, Tippi C. MacKenziea,⁎
aDivision of Pediatric Surgery and Fetal Treatment Center, Department of Surgery, University of California,
San Francisco, CA, USA
bBlood Systems Research Institute, San Francisco, CA, USA
cPerinatology Research Branch, NICHD/NIH/DHHS, Bethesda, MD, and Detroit, MI, USA
Received 18 February 2012; accepted 5 March 2012
Background/Purpose: Bidirectional trafficking of cells between the mother and the fetus is routine in
pregnancy and a component of maternal-fetal tolerance. Changes in fetal-to-maternal cellular trafficking
have been reported in prenatal complications, but maternal-to-fetal trafficking has never been studied in
the context of fetal intervention. We hypothesized that patients undergoing open fetal surgery would
have altered maternal-fetal cellular trafficking.
Methods: Cellular trafficking was analyzed in patients with myelomeningocele (MMC) who underwent
open fetal surgical repair (n = 5), patients with MMC who had routine postnatal repair (n = 6), and
healthy control healthy patients (n = 9). As an additional control for the fetal operation, trafficking was
also analyzed in patients who were delivered by an ex utero intrapartum treatment procedure (n = 6).
Microchimerism in maternal and cord blood was determined using quantitative real-time polymerase
chain reaction for nonshared alleles.
Results: Maternal-to-fetal trafficking was significantly increased in patients who underwent open fetal
surgery for MMC compared with healthy controls, patients who underwent postnatal MMC repair, and
patients who underwent ex utero intrapartum treatment. There were no differences in fetal-to-maternal
cell trafficking among groups.
Conclusion: Patients undergoing open fetal surgery for MMC have elevated levels of maternal
microchimerism. These results suggest altered trafficking and/or increased proliferation of maternal cells
in fetal blood and may have important implications for preterm labor.
© 2012 Elsevier Inc. All rights reserved.
⁎Corresponding author. Division of Pediatric Surgery/Fetal Treatment Center, Broad Center for Regeneration Medicine and Stem Cell Research, University
of California, Box 0570, San Francisco, CA 94143-0570. Tel.: +1 415 476 4086; fax: +1 415 476 2314.
E-mail addresses: email@example.com (P. Saadai), firstname.lastname@example.org (T.C. MacKenzie).
0022-3468/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
Journal of Pediatric Surgery (2012) 47, 1089–1094
Maternal-fetal cellular trafficking (MFCT) is the bidirec-
tional passage of cells across the placenta that results in the
presence of fetal cells in mothers [1-4] and of maternal cells
in offspring [5,6]. Increased amounts of fetal cells and cell-
free DNA have been seen in maternal serum after fetal
intervention  and may be a marker for preterm labor (PTL)
[8,9]. However, trafficking in the other direction (maternal
into fetal) is less well understood and has not been
investigated in the context of pregnancy complications.
Normal cellular trafficking in pregnancy may be a
component of maternal-fetal tolerance. It has recently been
reported that the presence of maternal cells in fetuses
(“maternal microchimerism”) may lead to the formation of
fetal regulatory T cells, which protect against an immune
response against the mother . Thus, alterations in
trafficking may be related to the breakdown of tolerance
between the mother and the fetus. Because PTL, a possible
consequence of such a breakdown in tolerance, remains the
Achilles, heel of fetal intervention , it is important to
We have previously described increases in maternal-to-
fetal cellular trafficking after fetal intervention in mice, with
particular increases in maternal T cells found in fetal blood
after allogeneic hematopoietic stem cell transplantation .
However, maternal microchimerism patterns after fetal
intervention in humans have not been studied. We examined
patients undergoing fetal surgery for repair of myelomenin-
gocele (MMC) to test the hypothesis that cellular trafficking
would be altered after open fetal surgery. We chose to study
patients with MMC because they are free of other underlying
hemodynamic or hematologic abnormalities that may affect
trafficking. In addition, because it is a nonlethal disease,
there is a control group of patients with the same disease who
do not undergo fetal intervention. We report that maternal
microchimerism is significantly increased in patients under-
going fetal MMC repair compared with healthy controls.
1. Materials and methods
This study was approved by the University of California,
San Francisco institutional review board (#10-00350).
Informed consent was obtained from all participants.
1.1. Cohorts and controls
Women carrying fetuses with MMC and healthy term
controls were prospectively recruited to participate in this
study between 2009 and 2011. Patients with MMC were
concurrently enrolled in the recently published Management
of Myelomeningocele Study randomized controlled trial 
and, thus, underwent either open hysterotomy fetal surgery
with subsequent cesarean delivery (“prenatal MMC” group)
or planned cesarean delivery with postnatal repair (“postnatal
MMC” group) at the University of California, San Francisco.
Given this delivery method, healthy-term pregnancies that
underwent planned cesarean delivery without the onset of
labor were included as controls. Healthy controls were also
included from the Perinatology Research Branch in Detroit,
MI, as part of an ongoing collaboration. As a control for
trafficking during an operation on placental support, 6
patients who underwent an ex utero intrapartum treatment
(EXIT) procedure  for various fetal anomalies were also
examined. Medical records were reviewed for maternal
history, operative reports, and perinatal course.
1.2. Fetal interventions
Open fetal surgery for repair of MMC was performed as
detailed in Adzick et al . Ex utero intrapartum
treatment procedures were performed as previously de-
scribed , with maternal general anesthesia to maximize
1.3. Sample collection
Cord blood samples for all infants were obtained at the
time of delivery, after cleaning the umbilical cord with
alcohol to avoid contamination with maternal blood.
Maternal blood was collected within 24 hours of delivery.
All blood samples were initially collected in EDTA-
containing tubes, and an aliquot of whole blood was frozen
for polymerase chain reaction (PCR) analysis. Blood
collected from Detroit was shipped on the day of delivery
by overnight mail on ice and processed upon arrival. All
blood processing was performed within 36 hours of delivery,
and samples were stored at −80°C.
1.4. Quantitative real-time PCR
Researchers blinded to patient groups quantified maternal
and fetal microchimerism using a quantitative reverse
transcriptase PCR assay that has been previously validated
 and used in the quantification of maternal blood in fetal
samples . Briefly, paired maternal and cord blood samples
were first genotyped for 12 HLA-DR and 12 In-Del alleles to
determine nonshared (“informative”) alleles between the
mother and the fetus after extracting genomic DNA. The
presence of microchimeric cells was then determined by
amplifying for the nonshared maternal alleles in cord blood or
also amplified with primers specific for HLA-DQ-α to
determine the concentration of total genomic DNA in each
specimen. The concentration of total DNA and minor-type
DNA (resulting from microchimerism) was calculated by
comparing the cycle threshold of the sample to those from
parallel amplifications of 10-fold serial dilutions of standards
individuals) and true positive (samples with a known
concentration of spiked cells) were consistently evaluated
1090P. Saadai et al.
correctly (data not shown). The lower limit of detection of this
depending on the DNA input and primer pair .
1.5. Statistical analysis
Pairwise comparisons were performed using the Mann-
Whitney U test for nonparametric or t test for parametric
data. Group-wise comparisons were performed using the
Kruskal-Wallis 1-way analysis of variance by ranks with a
Dunn post hoc test. A P value of less than .05 was considered
statistically significant. Statistics were performed using
Prism 5.0 (GraphPad Software, Inc, La Jolla, CA).
2.1. Demographic and operative characteristics
Relevant demographic and operative characteristics for
the groups are summarized in Table 1. Cell trafficking data
were analyzed, for a total of 26 pregnancies. Nine were
healthy patients who had cesarean deliveries at term without
labor. Eleven patients had fetal MMC, of whom 5 underwent
open fetal surgery at 24 ± 1.3 weeks and 6 underwent
postnatal surgery after planned delivery at 37 weeks. Six
patients underwent EXIT procedures for the following
indications: cervical teratoma (n = 1), congenital high-airway
GA at minimally invasive
GA at open fetal
28 ± 6.5
29 ± 5.5
25 ± 1.5
26 ± 2.8
27 ± 0.7
24 ± 1.3
36.5 ± 7.8⁎
131 ± 29
39 ± 0.9
32 ± 3.9
37 ± 0.9
34 ± 2.5⁎⁎
Values expressed as mean ± standard deviation. GA, gestational age; NA, not applicable.
⁎ P b .01 compared with operative time for open fetal surgery by t test.
⁎⁎P = .02 vs postnatal MMC, P b .01 vs healthy control.
for all groups, P = .01. *P b .05 by pairwise comparison using Mann-Whitney; **P b .05 by Kruskal-Wallis with Dunn post hoc comparison).
Maternal-to-fetal cellular trafficking could not be analyzed in 1 healthy patient because of the absence of nonshared alleles on PCR.
Maternal microchimerism. The percentage of maternal cells in cord blood was significantly different among groups (Kruskal-Wallis
1091 Alterations in MFCT after fetal surgery
obstructive syndrome (n = 1), sacrococcygeal teratoma (n =
2), and tracheal occlusion for congenital diaphragmatic
hernia (n = 2). Of note, 4 of the EXIT patients underwent
single-port minimally invasive fetal interventions during
pregnancy: 1 patient with sacrococcygeal teratoma under-
of 25 5/7 weeks (EXIT at 26 6/7 weeks), 1 patient with
5/7 weeks), and the 2 patients with congenital diaphragmatic
hernia underwent tracheal balloon insertion at 27 weeks
(EXIT at 29 and 36 weeks, respectively).
There were no differences in maternal age or infant sex
among groups. As expected, the prenatal MMC surgery
group delivered significantly earlier than both the postnatal
MMC (P = .02) and healthy control groups (P b .01) despite
routine postoperative tocolysis. The onset of PTL after open
fetal surgery was also reported in the results of the larger trial
comparing surgical outcomes in these patients . Opera-
tive time for prenatal MMC repair was significantly longer
than for EXIT procedure, where EXIT operative time was
defined as surgical start until delivery of the fetus (P b .01).
2.2. Cellular trafficking
Maternal-to-fetal trafficking (maternal microchimer-
ism). Informative (nonshared) alleles were identified in
25 of 26 samples (Fig. 1). Maternal microchimerism was
detectable in the 6 of 9 healthy control patients
undergoing normal-term cesarean delivery and undetect-
able in 2 healthy patients (median ± interquartile range,
0.0015 ± 0.015%); microchimerism could not be evalu-
ated in 1 healthy patient because of the lack of an
informative allele. These values were consistent with the
largest published series of cord blood microchimerism .
Maternal cells were detected in all 6 patients with MMC
undergoing postnatal repair (0.0145 ± 0.029%). Maternal
microchimerism was significantly increased in the 5
patients with MMC who had fetal surgery (0.2377 ±
0.099%, P b .05 vs postnatal MMC by Mann-Whitney
test and P b .05 compared with healthy controls and with
those who underwent EXIT procedures by Kruskal-Wallis
with Dunn posttest). Interestingly, the highest level of
MFCT (3.45%) was detected in a patient who underwent
an open fetal surgery whose postsurgical course was
complicated by prolonged oligohydramnios and recurrent
contractions with threatened PTL that continued for 1
month after her surgery. Maternal microchimerism was
low in 5 EXIT patients and undetectable in 1 (0.0080 ±
Fetal-to-maternal trafficking (fetal microchimerism).
Informative alleles were identified in 24 of 26 samples
(Fig. 2). There was no significant difference in the amount of
fetal-to-maternal cell trafficking among patient groups.
cellular trafficking could not be analyzed in 2 postnatal patients with MMC because of the absence of nonshared alleles on PCR.
Fetal microchimerism. The percentage of fetal cells in maternal blood was not significantly different among groups. Fetal-to-maternal
1092 P. Saadai et al.
This is the first study to examine maternal-to-fetal cell
trafficking in the context of fetal intervention. Although the
sample size is small, our results suggest that maternal
microchimerism is increased in patients undergoing open
fetal surgery followed by subsequent cesarean delivery but
not in patients who undergo fetal surgery during birth while
still on placental support (EXIT procedure).
The finding of increased maternal cells in fetuses that
have undergone open fetal surgery suggests that cellular
trafficking is altered after fetal surgery. An alternative
explanation, however, is that fetal surgery prompts the
proliferation of maternal cells that have already crossed—
perhaps as a result of the inflammatory signals that are
triggered after surgery. We examined MFCT in patients who
underwent EXIT procedures to determine whether undergo-
ing surgery while on placental support leads to immediate
leakage of maternal cells into the fetus; our results suggest
that changes in MFCT are more delayed. The continued
gestational period that follows prenatal MMC surgery but not
EXIT procedure may allow time for the activation of cellular
mechanisms that regulate trafficking and/or the proliferation
of trafficked maternal cells. Because several of the EXIT
patients had minimally invasive procedures before the EXIT,
our data also suggest that open hysterotomy fetal surgery
leads to more alterations in maternal microchimerism
compared with fetoscopy or percutaneous interventions.
In this study, we have not defined whether any particular
maternal cell types are preferentially recruited or proliferated
after fetal surgery. Our animal data suggest that granulocytes
are the predominant cell type in normal trafficking, with an
increase in T-cell trafficking after cellular transplantation
. Although this human study was performed with whole
blood, it would be possible to perform quantitative reverse
transcriptase PCR on sorted cells to determine if micro-
chimerism is increased in particular cell populations and
whether particular chemokines recruit distinct cell types
across the placenta.
Fetal-to-maternal cellular trafficking was not increased
in our study. This finding may be caused by the difficulty
in detecting small changes in levels of fetal cells in the
larger maternal blood volume or because trafficked fetal
cells may have diminished survival in maternal blood.
Another explanation may be that signals leading to
trafficking across the placenta are unidirectional and
governed by particular chemokines, rather than a general
“leakiness” of the placenta. An increase in cell-free fetal
DNA has been previously reported after less invasive fetal
interventions such as with laser coagulation for twin-twin
transfusion syndrome . It is possible that a surgical
intervention focused on the placenta leads to more
perturbations in cell trafficking, compared with the
nonplacental operations in our study.
Maternal-fetal cellular trafficking is a component of
maternal-fetal tolerance, and it is possible that alterations in
trafficking perturb this balance, resulting in PTL. In this
study, the prenatal MMC group with higher trafficking was
born at an earlier gestational age, but our study does not
establish a causal relationship between trafficking and PTL.
In addition, higher trafficking was not seen in several
fetuses that were delivered preterm via EXIT, indicating
that there is no absolute correlation between prematurity
and increased microchimerism. We are currently studying
cellular trafficking in patients undergoing PTL secondary to
nonsurgical causes as well as whether trafficking alters
immune reactivity between the mother and the fetus to
further characterize the relationship between trafficking and
PTL. A careful analysis of the specific types of cells that
traffic and the signals that promote their migration may
help determine if there is a causal relationship between
trafficking and PTL and may uncover strategic targets for
the prevention of preterm birth.
The long-term clinical significance of increased maternal
microchimerism in patients is unknown. Increased maternal
microchimerism has been described in patients with a variety
of diseases such as type 1 diabetes mellitus , neonatal
lupus syndrome–congenital heart block , and biliary
atresia [19-22]. However, it is unknown whether these
increased maternal cells in the offspring directly contribute to
the etiology of neonatal disease or are simply proliferating in
response to injury. Microchimerism may be tolerizing or
sensitizing to noninherited maternal antigens [23-26] and
may, therefore, have implications for graft tolerance in some
transplantation settings such as living-related bone marrow
 or liver transplantation . Thus, the study of the
effects of fetal intervention on maternal microchimerism may
have vital clinical significance for prenatally treated diseases
that may require postnatal organ transplantation, such as in
patients with posterior urethral valves.
Prenatal surgery for MMC is the most common open
fetal intervention performed worldwide  and is the only
open fetal surgery that has demonstrated efficacy in a
randomized controlled trial . Given the anticipated
expansion of open fetal surgery following the promising
results of the Management of Myelomeningocele Study
trial, our finding of increased maternal microchimerism
with this approach may have clinical implications for the
future health of these patients.
We thank the physicians and nurses at the UCSF Labor
and Delivery Unit and the Fetal Treatment Center for their
assistance with sample collection, Dr. Qizhi Tang for helpful
discussions, and our patients for their gracious participation
in this research project. This work was supported by a March
of Dimes Basil O'Connor Award (TCM), grants from the
California Institute for Regeneration Medicine (TCM and
AN), and the National Heart, Lung, and Blood institute
1093 Alterations in MFCT after fetal surgery
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Dr. Eugene Kim (Houston, TX): As an additional control, did
you ever get to study preterm babies that did not have any
Dr. Saadai (Response): That's an excellent question. We are
currently in the process of determining whether or not
this relationship with trafficking and PTL actually exists
in the patients with PTL independent of fetal surgery.
Dr. Tracy Grikscheit (Los Angeles): To look further into
your hypothesis that perhaps minimally invasive fetal
surgery might result in less trafficking, can you draw any
conclusions from the data that you already have in terms
of hysterotomy size or how invasive or you could
classify the open surgery?
Dr. Saadai (Response): It would certainly be interesting to
examine those factors. Our sample size precluded us
from performing such an analysis for this study but it is
certainly a question worth pursuing in the future.
1094P. Saadai et al.