Journal of Reproductive Immunology 108 (2015) 123–135
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Journal of Reproductive Immunology
journal homepage: www.elsevier.com/locate/jreprimm
Review
Treatment with granulocyte colony-stimulating factor in
patients with repetitive implantation failures and/or
recurrent spontaneous abortions
Wolfgang Würfel ∗
KCM (Kinderwunsch Centrum München) – Fertility Center Munich, Lortzingstr. 26, D-81241 München (Munich), Germany
a r t i c l e
i n f o
Article history:
Received 8 October 2014
Received in revised form 7 January 2015
Accepted 27 January 2015
Keywords:
Granulocyte colony-stimulating factor
(G-CSF)
Recurrent spontaneous abortion (RSA)
Repetitive implantation failure (RIF)
a b s t r a c t
Granulocyte colony-stimulating factor (G-CSF) belongs to the family of colony-stimulating
factors (CSF). As the name suggests, it was initially identified as being able to target and
influence granulopoiesis, but was soon shown to be a ubiquitous growth factor, with synthesis and receptors, such as the related granulocyte macrophage colony-stimulating factor
(GM-CSF), which is found in a wide variety of tissue types, including the organs and cell
populations involved in reproduction. It must now be assumed that both G-CSF and GMCSF control, or play a role in controlling, key processes in oocyte and sperm maturation,
endometrial receptivity, implantation, and embryo and fetal development, possibly extending to birth. The following article offers an overview of the current findings with regard
to animal experimental studies, initial clinical applications in reproductive medicine, and
potential risks.
© 2015 Elsevier Ireland Ltd. All rights reserved.
1. Historical background
The identification of colony-stimulating factors (CSF) is
based on cell culture assays established by Metcalf, Sachs,
and their assistants in the mid-1960s for hematopoietic
progenitor cells (Metcalf, 1980; Sachs, 1987). Granulocyte
colony-stimulating factor (G-CSF), with its ability to differentiate myelomonocytic leukemia cells in mice, was the
first to be isolated (Burgess and Metcalf, 1980). The description of human G-CSF followed in 1985 (Nicola et al., 1985;
Welte et al., 1985), and it became clear that G-CSF and GMCSF were different cytokines (Metcalf, 1985; Sachs, 1987).
Mapping of the G-CSF gene and cDNA was performed
in 1986 (Souza et al., 1986; 174 amino acids). Nomura
et al. (1986) cloned further cDNA with 177 amino acids,
∗ Tel.: +49 89 244144 99/91; fax: +49 89 244144 41.
E-mail address: [email protected]
http://dx.doi.org/10.1016/j.jri.2015.01.010
0165-0378/© 2015 Elsevier Ireland Ltd. All rights reserved.
a less effective derivative originating from a tumor cell line
(CHU-2).
2. G-CSF and receptor
2.1. Structure of G-CSF and its receptor
Granulocyte colony-stimulating factor is coded by a single gene (Nagata et al., 1986), localized on chromosome 17
(q11-22) (LeBeau et al., 1987) and thus differing from other
growth factors such as GM-CSF, IL-3, IL-4, and IL-5 (chromosome 5) (Nicola, 1989). The gene comprises five exons
and has a length of approximately 2.3 kb; the splicing variants are encoded in tandem at the 5 terminus of the second
intron and are 9 bp apart (Nagata et al., 1986).
G-CSF acts via specific high-affinity receptors (Nicola
and Metcalf, 1985). Although four receptors were originally
assumed (Bazan, 1990; Fukunaga et al., 1990), there are in
fact two receptors (Larsen et al., 1990), one with 759 and
124
W. Würfel / Journal of Reproductive Immunology 108 (2015) 123–135
the other with 812 amino acids; their primary distinction
is at the carboxyl terminus. The gene is located on chromosome 17 (q11.2-12) (Tweardy et al., 1992; Tkatch and
Tweardy, 1993) and the molecular weight is approximately
19.06 kDa.
2.2. Function
The G-CSF receptor is part of the cytokine receptor
superfamily (Rapoport et al., 1992) and functions by means
of tyrosine phosphorylation and activation of members of
the Janus protein kinase family (Jak) (Shimoda et al., 1997),
particularly Jak1, Jak2, and Tyk2. In addition, there is an
activation pathway of the STAT family (signal transducers
and activators of transcription), primarily of STAT1, STAT5,
and STAT3 (Marino and Rogiun, 2008), and likewise for
mitogen-activated protein kinases (MAPK) such as p38 and
p44/42 MAPK (also Marino and Rogiun, 2008).
2.3. Tissue distribution of the G-CSF receptor
As the name suggests, receptors are found in many types
of myeloproliferative tissue and their cells, e.g., in monocytic cells such as macrophages, natural killer (NK) cells
(Miyama et al., 1998), and T cells (Franzke et al., 2003),
and also on platelets (Shimoda et al., 1993). Receptors can
also be found on cells of myeloproliferative or lymphoid
diseases (van de Gejin et al., 2003), and in many solid malignomas and their metastases (review in Liongue et al., 2009),
such as prostate (Savarese et al., 1998) and ovarian carcinoma (Savarese et al., 2001), and also on choriocarcinoma
cells (Vandermolen and Gu, 1996).
During maturation of the preovulatory follicle, G-CSF
receptor expression increases (Fujii et al., 2011); receptor
expression also takes place in luteinized human granulosa cells (Salmassi et al., 2004). G-CSF receptor expression
occurs in human endometrial cells throughout the cycle
at varying densities of expression (Vandermolen and Gu,
1996), influenced by estrogen (Zeyneloglu et al., 2013).
All receptors exist in the placenta (Larsen et al., 1990;
Fukunaga et al., 1990), and on the trophoblast (Uzumaki
et al., 1989). This, together with the synthesis of G-CSF itself
in both human and murine cyto- and syncytiotrophoblasts,
was later confirmed by other researchers (Saito et al.,
1994). The expression pattern evidently changes over the
course of pregnancy; the highest G-CSF receptor expression in the first trimester primarily occurs in the interstitial
cytotrophoblast and the decidua, declines over the second
trimester, and rises again in the third (McCracken et al.,
1999).
The human fetus possesses G-CSF receptors in virtually
all tissue types, particularly renal and gastrointestinal tract
tissue (Calhoun et al., 1999, 2000), on hemopoietic and neural stem cells (Liu et al., 2009), and in various areas of the
brain, particularly the radial glia, during development of
the central nervous system (Kirsch et al., 2008). The picture
is similar in adult humans: G-CSF (and GM-CSF) receptors
are found, for example, on endothelial cells (Bussolino et al.,
1989) and fibroblasts (Miyama et al., 1998), in the liver
(humans, rats, and mice) (Fang et al., 2013; Meng et al.,
2012), in the prostate (Savarese et al., 1998), in the brain
(Ridwan et al., 2014), and on neurons (Schneider et al.,
2005).
2.4. Cellular synthesis of G-CSF
Natural killer cells, particularly uterine NK cells
(CD56bright ), can synthesize CSFs including G-CSF (Sharma
and Das, 2014), as was demonstrated as early as
1989 (Cuturi et al., 1989). For a current review, see
Niedzwiedzka-Rystwej et al. (2012). Further immunocompetent cells include macrophages/monocytes (Vellenga,
1996; Aoki et al., 2000), T cells (Nioche et al., 1988; Demetri
and Griffin, 1991; Moore, 1991; Matsushita et al., 2000),
and dendritic cells (Cheknev et al., 2014).
Granulocyte colony-stimulating factor is synthesized by
granulosa cells and luteal cells (Yanagi et al., 2002; Salmassi
et al., 2004; Fujii et al., 2011), a process that increases, particularly during the preovulatory phase (Yanagi et al., 2002;
Fujii et al., 2011). In general, epithelial apical cells in the
female reproductive tract (fallopian tubes, endometrium,
endocervix) synthesize G-CSF in in vitro cell cultures
(Watari et al., 1994; Fahey et al., 2005; Braunstein, 2007);
synthesis has also been demonstrated for endometrial cells
in vivo (Vandermolen and Gu, 1996). Synthesis is positively
regulated by estrogens (Zeyneloglu et al., 2013) and progesterone, IL-1␤, and IL-6, while hypoxia causes the synthesis
rate to fall, particularly in stromal cells (Braunstein, 2007).
Granulocyte colony-stimulating factor is synthesized in
decidual cells (Duan, 1990; Shorter et al., 1992) and in the
chorionic villous trophoblast (Shorter et al., 1992), but not
in the cytotrophoblast. The synthesis rate for both cell types
is significantly higher in the first trimester than at term. Li
et al. (1996) found that the human placenta in vivo contains
little or no G-CSF mRNA (admittedly not necessarily a contradiction), but also that under IL-1a stimulation, the term
placenta has a high capacity for synthesizing G-CSF. They
also found that placental G-CSF synthesis does not affect
fetal granulocytopoiesis.
Further sources are fibroblasts (Fibbe et al., 1988;
Vellenga, 1996), including dental pulp fibroblasts (Sawa
et al., 2003), mesothelial cells (Lanfrancone et al., 1992),
neuronal stem cells (Schneider et al., 2005), and (fetal) villus enterocytes (Calhoun et al., 2000).
Furthermore, neoplastic G-CSF synthesis has been
known for a relatively long time for various tumor entities (Tsukuda et al., 1993; Joshita et al., 2009), including
different types of leukemia (Matsushita et al., 2000;
Rodriguez-Medina et al., 2013).
3. Physiological significance of G-CSF
As the name implies, CSFs play a significant role in a
wide variety of granulo- and myeloproliferative processes.
It has long been known that CSFs perform further physiological functions, including in the murine implantation
process (for an overview see Pampfer et al., 1991).
Granulocyte colony-stimulating factor is supposed to be
a major co-factor in the mediation of the human ovulation
process (Makinoda et al., 2008). The authors thus discuss
potential clinical applications of G-CSF, e.g., in cycle disorders or luteinized unruptured follicle (LUF) syndrome.
W. Würfel / Journal of Reproductive Immunology 108 (2015) 123–135
Granulocyte colony-stimulating factor promotes the
proliferation of chorionic cells (Duan, 1990). During pregnancy, concentrations are higher than in nonpregnant
women (Umesaki et al., 1995), in particular, shortly before
the onset of labor (Raynor et al., 1995; Bailie et al., 1994),
although this is not undisputed (Calhoun et al., 2001a,b).
Correlation between the neutrophil counts and G-CSF concentrations is claimed to be particularly significant during
the third trimester (Umesaki et al., 1995). Here too, results
differ (Matsubara et al., 1999), although these authors find
a significant connection between G-CSF serum concentration and pre-eclampsia with regard to birth weight and
diastolic and systolic blood pressure. G-CSF is present in the
amnion (Raynor et al., 1995). Amniotic infection syndrome
(AIS) causes the concentration to rise (Raynor et al., 1995),
in maternal serum (Boggess et al., 1997) and neonatal urine
too, while receptor expression in the placenta increases
simultaneously (Calhoun et al., 2001a,b).
Granulocyte colony-stimulating factor promotes the
proliferation of neural stem cells (Liu et al., 2009) and is
a promoter of neurogenesis (Schneider et al., 2005) and an
apoptotic antagonist, which is also described for JEG-3 cell
lines (Marino and Rogiun, 2008).
Granulocyte colony-stimulating factor causes concentrations of regulatory T cells to rise (Rutella et al., 2002,
2005; Rutella, 2007), as has also been described in patients
with spontaneous abortions (Sbracia and Scarpelini, 2012),
and the activation of dendritic cells (for an overview see
Adusumilli et al., 2012). G-CSF reduces the toxicity of
NK cells (Taga et al., 1993) in various ways: by reducing the generation of NK cell progenitors (CD34+ /CD2+ ,
CD34+ /CD7+ , CD 34/CD10+ ) (Miller et al., 1997), by reducing
the expression of activating receptors (e.g., NKG2D receptor) and inhibitory receptors (e.g., KIR2DL1 or KIR2DL2),
by reducing the cytokine synthesis of interferon-␥, TNF-␣,
GM-CSF, IL-6, and IL-8 (Schlahsa et al., 2011), by lowering
the secretion of IFN-␥ and sFasL (Su et al., 2012), and by
reducing aggression toward K562/K562:HLA-E*01:03 target cells, both in vitro and in vivo (Kordelas et al., 2012).
At the same time IL-4 synthesis in CD4+ and CD8+ lymphocytes is increased, causing the Th1/Th2 ratio to shift in favor
of the Th2 response (Sloand et al., 2000).
Both G-CSF and GM-CSF cause increased migration and
proliferation of endothelial cells (Bussolino et al., 1989).
An effect of G-CSF that is more pathological than physiological is its ability – when synthesized in malignomas – to
stimulate tumor growth, demonstrated for cases including
head and neck carcinoma (Tsukuda et al., 1993), cervix carcinoma (Kyo et al., 2000), and bladder cancer (Chakraborty
and Guha, 2007), and to promote metastases (Kowanetz
et al., 2010).
4. Animal experimental studies with regard to
reproduction
The presence of G-CSF receptors can be proved in
myeloid cells (CD11b), lymphoid cells (CD3, CD19), NK
cells, and dendritic cells (CD11c) of mice and murine
embryos, in addition to numerous other tissues including
liver (hepatocytes), cardiac muscle, kidney, brain, intestine
125
(ileum and colon), and vascular endothelial cells (Touw
et al., 2007: csf3r-Cre knock-in mice).
Granulocyte colony-stimulating factor-deficient mice
have fewer neutrophil granulocytes; they are viable and
can reproduce (Lieschke et al., 1994), albeit at reduced
fertility levels, but show a significantly higher rate of spontaneous abortions (in addition to lower life expectancy and
a tendency toward amyloidosis (Seymour et al., 1997)).
G-CSF has been proven to have an anti-abortive effect in
mice (Litwin et al., 2005). Under the influence of G-CSF
(like GM-CSF), preimplantation embryos in mice develop
a higher percentage of hatched blastocysts (Kim et al.,
2002). This also particularly applies to the implantation
rate at 10 pg/ml and 100 pg/ml (culture duration: 96 h). As
the number of expanded blastocysts did not increase, the
authors concluded that G-CSF (like GM-CSF) increases the
implantation capability of murine blastocysts. In addition,
it can be assumed that murine preimplantation embryos
must express G-CSF receptors. G-CSF stimulates the proliferation of porcine trophectoderm cells (Jeong et al., 2014).
Systemically applied G-CSF passes the placenta barrier
in rats and stimulates fetal myelopoiesis, although it evidently does not affect the thymus or liver (Medlock et al.,
1993). Placental passage has also been demonstrated in
mice; here, increased concentration in the fetus can be seen
only 30 min after a single dose of 50 ␮g/kg rh-G-CSF (with
a peak concentration after 2 h) (Calhoun et al., 2001a,b).
In cases of B streptococcal infection, the maternal application of G-CSF improves the survival rate of the offspring
(Novales et al., 1993).
High-dose G-CSF inducing peripheral leukocytosis
results in placental embolism and an increased rate of abortion in animal experiments (Keller and Smalling, 1993).
However, this only occurs at extremely high doses and only
in rabbits; it has not been observed in rats, mice or monkeys
(Okasaki et al., 2002).
In rats, G-CSF causes thickening of the endometrium,
principally after injury to the same (Zhao et al.,
2013). In diabetic rats (experimental diabetes mellitus),
intraperitoneal injection of G-CSF (100 ␮g/kg/day) reduced
endometrial gland degeneration and fibrosis and significantly reduced ovarian follicle degeneration, slowing the
decline of anti-Müllerian hormone (AMH) (Pala et al.,
2014).
In mice, G-CSF develops a cell-protective effect under
chemotherapy with alkylating agents, and postpones
the onset of ovarian insufficiency (Skaznik-Wikiel et al.,
2013a,b). Again, in mice, G-CSF together with vasoendothelial growth factor (VEGF) reduces ischemic damage in
ovarian transplantation and results in significantly greater
preservation of primordial follicles (Skaznik-Wikiel et al.,
2011). The protective effect is claimed to be equal to that
under leuprolide (Skaznik-Wikiel et al., 2013a,b).
5. Clinical applications
5.1. G-CSF and oocyte maturation
A positive correlation between G-CSF concentration in
follicular fluid and IVF outcome was suspected as early as
2005 (Salmassi et al., 2005). Follow-up studies showed that
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W. Würfel / Journal of Reproductive Immunology 108 (2015) 123–135
Table 1
Results of our first study (2000): patients with more than four IVF/ICSI treatments not resulting in pregnancy or more than four unsuccessful ETs received
molgramostim, and, in a second series, filgrastim as a single shot. The age of all groups was between 37 and 38; all ETs were performed on day 2 (D + 2).
Number of patients
Number of embryo transfers (day 2)
Clinical pregnancies
Pregnancy rate/embryo transfer
Abortion rate (clinical pregnancies)
H-GM-CSF (molgramostim 300 ␮g)
No medication
Rh-G-CSF (filgrastim 34 mIU)
No medication
107
107
46
42.9%
9 (19.5%)
107
106
21
19.8%
2 (19.0%)
69
69
35
50.7%
7 (29%)
69
69
13
19.8%
2 (15.4%)
high concentrations of G-CSF increased the probability of
implantation and subsequent pregnancy (Frydman et al.,
2009; Lédée et al., 2008, 2010a,b, 2011a,b,c). This evidently
applies only to G-CSF; no such correlation is shown by other
growth factors and cytokines (Osipova et al., 2009; Piccinni
et al., 2009). According to a more recent publication from
the same working group, oocytes from follicles with a concentration of >30 pg/ml show the highest probability (class
I), while those with a concentration of 18.4–30 pg/ml show
mid-range probability (class II), and oocytes from follicles
with a concentration of <18.4 pg/ml show low probability
(class III) of resulting in pregnancy in an assisted reproduction technology (ART) program (embryo transfer [ET]:
day 3) (Lédée et al., 2013b); average implantation rates
were 36% (class I), 16.6% (class II), and 6% (class III). While
the morphology in these cases does not necessarily correlate with the G-CSF concentration (Lédée et al., 2012),
excellent embryo morphology in tandem with a high GCSF concentration results in the highest implantation rates
(54%) (Lédée et al., 2012). Measurement of G-CSF concentration in follicular fluid evidently also enables a prognosis
of the later pregnancy rate (per oocyte harvested) to be
made, even for patients with severe ovarian insufficiency
(AMH < 0.1 ng/ml) (Lédée et al., 2013a): conception was
zero among patients with class III oocytes, while patients
with follicular G-CSF concentrations >18 pg/ml showed a
pregnancy rate of 35%. A corresponding testing system for
follicular G-CSF concentration in ART treatment is now
available commercially under the name DiafertTM .
The correlation between high G-CSF concentration in
follicular fluid and favorable pregnancy prognosis in ART
treatment would suggest systemic administration of G-CSF
in the follicular phase. While no studies on this theme exist
as yet, we are currently performing a pilot study (with
13 mIU filgrastim every second day). Initial results indicate
an improvement in the pregnancy rate.
A single dose of rh-G-CSF (lenograstim, 100 mg) 48 h
before the administration of HCG results in a significant
reduction in LUF syndrome in clomiphene citrate (CC)
cycles (Fujii et al., 2013; comparable: Makinoda et al., 2012;
Tomizawa et al., 2010, 2011). This procedure may enable
roughly 90% of all LUF syndromes to be avoided (Makinoda
et al., 2010).
5.2. G-CSF and its influence on early embryonic
development and implantation (in ART treatment), in
particular in patients with repetitive implantation failures
A randomized, double-blind, placebo-controlled, multicenter study confirmed the favorable effect of rh-GM-CSF
– closely related in many ways – in culture medium
on pregnancy and abortion rates (Ziebe et al., 2013): in
1149 ETs, the implantation rate rose significantly from 20%
to 23.5%, and the birth rate from 24.1% to 28.9%. The culture
medium is now commercially available under the name
EmbryogenTM . A patent has also been requested for culture medium with added 0.5 ng rh-G-CSF/ml (Scarpellini
and Sbracia, 2014).
In 2000, we reported our experiences with h-GM-CSF
(molgramostim) and rh-G-CSF (filgrastim) in patients with
repetitive implantation failure (RIF), administered as a
single shot at the time of ET (Würfel, 2000). Results are
depicted in Table 1. Administration of human-derived
GM-CSF was relatively frequently accompanied by undesired side-effects such as high blood pressure and chest
tightness, which was not the case with filgrastim.
In 2010, we reported on the continuous application of
rh-G-CSF (13 mIU lenograstim every third day after ET)
in patients with long-term sterility and/or five unsuccessful ART treatments, and deficiency in activating killer
immunoglobulin-like receptors (KIR) (see Hiby et al., 2008,
2010). The pregnancy rates achieved were significantly
higher than the pregnancy rates of our ART program for
both day 2 ETs; pregnancy rate: (42% per ET) and day 5
ETs (73.8%). Table 2 shows the preliminary results from an
ongoing study. The study is randomized but not blinded.
rh-G-CSF is applied after the sixth cycle in total (including cycles performed elsewhere). Significance (p < 0.5) is
currently seen in many cycles, although not in all.
In 2012, Scarpellini and Sbracia reported on a randomized controlled study, also of 109 RIF patients. RIF
was described as the status after a minimum of three IVF
attempts with transfer of a minimum of seven embryos of
good morphology; the maximum age of the patients was
39 and they were free from systemic disorders. From the
time of ET (presumably performed on day 2) they received
a daily dose of 60 mg G-CSF (no more specific details of
the compound were given), which was continued for a further 40 days after a positive pregnancy test. The (clinical)
pregnancy rate was 43.1% per ET in the G-CSF group (58
patients) and 21.6% in the placebo group (saline injections)
of 51 patients, resulting in a highly significant difference
(p < 0.001). No undesirable side-effects were reported. Similar results were reported by Gnoth (2014) in 46 RIF patients
(at least three failed ART attempts) with a cumulative pregnancy rate of 46% under G-CSF.
5.3. G-CSF in patients with recurrent spontaneous
abortions
Scarpellini and Sbracia (2009) reported the results
of a randomized placebo-controlled study of recurrent
W. Würfel / Journal of Reproductive Immunology 108 (2015) 123–135
127
Table 2
Preliminary results of the ongoing study in our IVF/ICSI program for patients with repetitive implantation failures (RIF; results up to December 2013). G-CSF
is applied with the sixth cycle. Depending on the number of fertilized oocytes, ET was performed after 2–3 days (D + 2/+3) or after 5 days (D + 5). Number
of treated patients D + 2/+3: 191, number of patients D + 5: 86.
No medication, D + 2/+3
Rh-G-CSF (filgrastim) D + 2/+3
No medication D + 5
Rh-G-CSF (filgrastim) D + 5
Age (years)
Number of embryos transferred
37.0
2.1
37.1
2.0
38.1
1.9
37.9
2.0
Pregnancy-rate/ET
1st cycle
2nd cycle
3rd cycle
4th cycle
5th cycle
6th cycle
7th cycle
8th cycle
9th cycle
10th cycle
29.4%
39.6%
33.7%
23.1%
22.2%
17.9%
13.7%
10.2%
12.0%
12.7%
–
–
–
–
–
56.0%
31.3%
33.2%
20.4%
13.7%
43.1%
52.4%
50.4%
33.3%
31.1%
29.1%
30.0%
24.1%
20.1%
21.9%
–
–
–
–
–
69.1%
39.3%
20%
59.9%
49.6%
spontaneous abortion (RSA) patients. Inclusion criteria were women’s age <39 years, more than four
previous spontaneous abortions, failed previous treatment with immunoglobulins (IVIG), normal karyotype,
no uterine defects or malformations (ultrasound and/or
hysteroscopy), no infections, e.g., chlamydia, CMV, toxoplasmosis, herpes, no thrombophilia, no antiphospholipid
antibodies and no other autoimmune antibodies, no insulin
resistance, and no thyroid disorders. Treatment began on
the sixth day after ovulation with administration of 1 ␮g/kg
rh-G-CSF daily (filgrastim).
Demographic data and results are depicted in Table 3.
The birth rate in the G-CSF group showed a highly significant increase up to the same level as primiparous women
without RSA problems. The obstetric outcome for both
groups was similar, as was the abortion rate; no malformations were reported. Noteworthy is the significant increase
in HCG levels in the G-CSF group, as they were an average
of 30% higher than in the control group in the ninth week
of gestation.
Table 4 shows the results of the study by Santjohanser
et al. (2013) of RSA patients in an IVF/ICSI program. This
study had a third arm in which patients were given
low molecular weight (lmw) heparin, cortisone, and/or
immunoglobulin – in other words, drugs believed to reduce
abortion rates (Toth et al., 2010, 2011). In this group and
in the serum group, the pregnancy rate was significantly
lower than in the G-CSF group.
Evidence that G-CSF is able to lower abortion rates
has existed for some time, primarily derived from the
Severe Chronic Neutropenia International Registry (SCNIR).
For example, Boxer et al. reported in 2010 that patients
with neutropenia receiving 1.07 ␮g/kg/day of G-CSF during
pregnancy showed significantly fewer abortions – namely
5% compared with 32% in patients who did not receive GCSF.
Table 5 shows the results of our treatment in RSA
patients. Preliminary diagnosis was made according to
the relevant guidelines (Toth et al., 2010, 2013), including
diagnosis for chromosomal aberrations, antiphospholipid
syndrome, coagulation disorders, or uterine malformations
– although antiphospolipid syndrome, for example, can
be successfully treated with a combination of G-CSF and
heparin (Hönig et al., 2010). Similar to Scarpellini and
Sbracia’s findings, patients with an average abortion rate
of 4.7 had received a variety of treatments in previous
pregnancies, including immunoglobulin in some cases. At
approximately 80% (78.1%) per commenced pregnancy, the
birth rate was similar to the results of Scarpellini and
Sbracia. Also similar to their results, we also registered
significantly higher levels of HCG, and therefore also progesterone (personal communications); between the tenth
and twelfth week of pregnancy, biometry almost regularly showed a CRL (crown rump length) around one week
further advanced (unpublished data); the gestational sacs
were always significantly enlarged (unpublished data).
The indication for administration of G-CSF was not
based on a diagnosis of exclusion, but on the following considerations and test results: It can be assumed that uterine
NK cells (uNK) in particular, with their KIR, enter a dialog with the conceptus and the trophoblast (Herrler et al.,
2003; Makrigiannakis et al., 2008). Disruption of implantation and development during pregnancy is the result
both of a lack of inhibitory interaction (Varla-Leftherioti
et al., 2004) and a lack of activating interaction (Hiby et al.,
2008, 2010). As G-CSF is a growth-promoting cytokine, we
regard disruption of activating interaction as an indication.
Patients lacking activating KIR actually benefit from G-CSF
(Würfel et al., 2010; Santjohanser et al., 2011), particularly
if 2DS3 and 3DS5 are lacking (Hirv and Würfel, 2010). While
we originally used KIR typing as the exclusive basis for
defining an indication, we now follow the results of Hiby
et al. (2008, 2010) more closely, seeing an indication for GCSF if there is a lack of activating KIR in combination with
(weak) paternal HLA-C2 groups or a corresponding high
haploidentity of the adult HLA complex of the couple (see
also Toth et al., 2011).
However, at present there is no precise definition of an
‘implantative G-CSF deficiency syndrome’ (‘IGDS’) and no
proper dosage-finding studies have been carried out.
5.4. G-CSF for thin endometrium
In 2012, Gleicher et al. reported cases of patients
with thin endometrium who had received G-CSF as an
intrauterine instillation. This flushing resulted in increased
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W. Würfel / Journal of Reproductive Immunology 108 (2015) 123–135
Table 3
Demographic data for women with unexplained recurrent miscarriage (RSA) and results if treated with rh G-CSF (filgrastim) or saline (placebo) (Scarpellini
and Sbracia, 2009).
Number of patients
Age (years) when pregnancy started
BMI: when pregnancy started
Smokers (more than 10 cigarettes per day)
Number of previous miscarriages
Gestational week of miscarriage
Number of live births (%)
Number of miscarriages (%)
Gestational week of miscarriage (mean ± SD)
Newborn weight (g) (mean ± SD)
Rh-G-CSF
Placebo
P value
35
34.9 ± 2.9
27.4 ± 1.9
1
5.5 ± 0.4
6.1 ± 1.2
29 (82.8%)
6 (17.2%)
6.0 ± 1.1
3050 ± 220 g
33
33.8 ± 2.9
33.8 ± 1.8
2
5.6 ± 0.3
6.4 ± 1.1
16 (48.5%)
17 (51.5%)
6.2 ± 1.0
3125 ± 240 g
0.0061
0.0061
0.6989
0.3098
Table 4
Results of the study by Santjohanser et al. (2013), concerning IVF/ICSI patients with RSA.
Age (years)
Number of previous cycles
Number of oocytes
Number of fertilized oocytes
Number of transferred embryos
Number of early miscarriages (anamnestic)
Number of late miscarriages (anamnestic)
Number of embryo transfers (ET)
Pregnancies (pregnancy rate per ET)
Clinical abortions
Rh-G-CSF (filgrastim)
No medication
Other medication (e.g., low
molecular weight heparin, ASS
(100 mg), prednisone/
dexamethasone, doxycycline)
37.6 ± 4.0
6.5
9.4 ± 5.6
5.7 ± 3.9
2.1
2.7 ± 1.3
0.2 ± 0
49
23 (46.9%)
7 (30.4%)
37.6 ± 4.4
5.3
9.6 ± 5.0
5.0 ± 3.4
2.5
0.9 ± 0.9
1.8 ± 1.0
33
8(24.4%)
4 (50%)
37.6 ± 4.4
6.0
7.8 ± 4.4
4.9 ± 3.4
2.4
0.6 ± 0.7
1.8 ± 1.0
45
12 (26.6%)
6 (50%)
endometrial thickness; all four patients received ET and
conceived. A subsequent noncontrolled cohort study confirmed the effect on the endometrium (Gleicher et al.,
2013). Kunicki et al. (2014) and Lucena and Moreno-Ortiz
(2013) came to the same conclusion with regard to PCO
syndrome.
The procedure used by Yu et al. (2014) for ETs with
cryopreserved preimplantation embryos was similar; they
instilled 100 ␮g/0.6 ml rh-G-CSF on the day of ovulation
induction with HCG or when progesterone application was
commenced. While they failed to prove an increased pregnancy rate per ET, the control groups showed a 69.4% and
Table 5
Gestational outcome of RIF and RSA patients up to May 2013. Included are the results of patients with recurrent abortions if ART treatment was necessary.
RIF patients
RSA patients
>37th week of gestation
301
265 (88.0%)
79 (29.6%)
49 (62.0%)
38 (67.4%)
186 (72.4%)
161
24
1
139 (86.3%)
308
283 (91.8%)
62 (21.9)
41 (66.1%)
25 (60.9%)
221 (78.1%)
219
2
None
190 (86.9%)
32nd–37th week of gestation
<32nd week of gestation
>37th week
19 (11.8%)
3 (1.9%)
3231 ± 1351 g
20 (9.0%)
11 (5.1%)
3412 ± 1512 g
32nd–37th week
<32nd week
2424 ± 578 g
1112 ± 227 g
1 acrocephaly; 1 omphalocele;
2 induced abortions (47, XX,
+21; 47, XY, +18); 1 born child
with 47, XX, +13
2206 ± 625 g
1080 ± 485 g
2 omphaloceles; 4 induced
abortions (47, XY, +21; 47, XX,
+21; 47, XY, +18; complex
malformation syndrome
without genetic abnormality)
1 intrauterine death, 18th
week of gestation, no cause
found
Clinical pregnancies
Data available
Clinical abortions
Parturitions
Singleton pregnancies
Twin pregnancies
Triplets
Time of delivery (singleton
pregnancies)
Mean birth weight (singleton
pregnancies)
Malformations/abnormal genetic
findings in pregnancy
Obstetrical complications
Tested genetically
Abnormal results
186 (72.4%)
1 child died of cerebral
hemorrhage (twin pregnancy)
W. Würfel / Journal of Reproductive Immunology 108 (2015) 123–135
48.8% rate of cycle cancelation compared with only 17.5%
in the G-CSF group, demonstrating a highly significant rise
in pregnancy rate per cycle started.
We have ourselves observed sustained endometrial
growth with the systemic administration of rh-G-CSF
(lenograstim, 13 mIU) every second or third day during the
follicular or stimulation phase (unpublished data).
5.5. Further clinical applications
The administration of G-CSF during pregnancy to reduce
the frequency of preeclampsia was patented in the USA in
2013 (Carter; US 8470775 B2). Further patents accordingly
refer, for example, to the use of G-CSF to reduce the abortion rate (US 8338373; Carter, 2009) or to reduce premature
birth (US 8481488, Carter, 2012). More patents concerning further indications for G-CSF applications are pending
from the same working group or inventor (Noratherapeutics, Carter et al.).
6. Risks
6.1. G-CSF and the induction of malignoma
A major indication for G-CSF is during or after
chemotherapy, i.e., the treatment of malignomas, particularly the prevention and treatment of febrile neutropenia
(FN) (Aapro et al., 2011; Falandry et al., 2010). Current
guidelines recommend the use of G-CSF where indications
are given and do not see a risk of tumoral progression
or lymphoproliferative disorder, or even the induction
of a second malignoma, e.g., breast cancer (DGGG, DKG,
2012), Hodgkin’s lymphoma (DKG, DKH, 2013), guidelines
of EORTC and ASCO (Aapro et al., 2011; Falandry et al.,
2010), of ESMO (Crawford et al., 2010), and of the North
Wales Cancer Network (2009). There is evidence that GCSF does not increase the percentage of circulating tumor
cells (CTC), a key factor in prognosis (SUCCESS Study) (Hepp
et al., 2009; Jenderek et al., 2009). However, G-CSF is not
wholly without effect on tumor development: in combination with chemotherapy, a significant rise in tumor
markers has been observed, and murine animal trials have
shown that G-CSF promotes at least tumor neoangiogenesis and counteracts the effect of chemotherapy to a certain
degree (Okazaki et al., 2006; Voloshin et al., 2011). In these
cases, however, the overall dose and duration were significantly higher than in the regimes applied in reproductive
medicine or bone marrow donation, although they were
significantly lower than in patients with chronic neutropenia.
Bone marrow donors generally receive rh-G-CSF after
the donation process. Large-scale follow-up investigations of healthy bone marrow donors treated for short
periods (3–15 days) with rh G-CSF (filgrastim; generally 13–16 ␮g/kg/day) did not show an increased risk of
myeloid leukemia and/or myelodysplasia over a followup observation period of 3–6 years (Anderlini et al., 2002;
Cavallaro et al., 2000). The same conclusion was reached by
an evaluation of the National Marrow Donor Program Registry (Pulsipher et al., 2009), in which 2408 donors were
recorded over a period of up to 99 months. In 2009, Halter
129
et al. reported an incidence of 8 new malignant hematological cases out of 27,770 healthy bone marrow donors and 12
such cases out of 23,254 peripheral blood stem cell (PBSC)
donors. In both groups the incidence of malignancies was
below the expected level for the corresponding age groups.
However, it may be assumed that continuous and, in
some cases, high-dosage administration of G-CSF over
a period of years might result in the development
of myelodysplasia syndrome (MDS) or acute myeloid
leukemia (AML). Freedman et al. (2000) reported an incidence of 9% in SCNIR data over a 13-year period of
observation. Current findings (Rosenberg et al., 2010) show
an increased annual risk of 2.3% after 10 years and a cumulative risk of approximately 22% after 15 years. Similar
observations have been made in recipients of stem cells,
albeit with a lower cumulative risk (Falkenburg, 2001). The
issue of whether this is actually related to continuous and,
in some cases, high-dosage administration of G-CSF, or is
the result of a predisposition from the underlying disorder,
e.g., the neutropenia, is still unclarified (Avalos et al., 2011).
A further issue under discussion has been whether longterm continuous administration can result in increased
rates of cellular aneuploidy (Freedman et al., 2000). More
recent data show that this is not the case (Hirsch et al.,
2011).
6.2. Administration of G-CSF during pregnancy
As already stated, systemically administered G-CSF
can pass the placental barrier in rats and in humans
(Medlock et al., 1993). The authors speculate that this might
improve neonates’ defenses against infection. In fact, a single dose of G-CSF (25 ␮g/kg) in 26 women with a history
of premature births resulted in increased fetal synthesis
of neutrophil granulocytes and lower neonate morbidity
(Calhoun and Christensen, 1998); no undesirable sideeffects were observed. This is confirmed by several case
reports of women with severe neutropenia who received
injections of G-CSF several weeks before birth and gave
birth to healthy babies (Kaufmann et al., 1998; Abe et al.,
2000; Sangalli et al., 2001).
In addition, there are numerous reports of pregnant
patients who have received G-CSF, primarily to support
chemotherapy (Cardonick et al., 2012), but also to mobilize
autologous stem cells during chemotherapy in the second
and/or third trimester (Cavenagh et al., 1995; Lin et al.,
1996; Reynoso et al., 1987; Aviles and Neri, 2001; Siu et al.,
2002). None of the authors noted congenital malformations or other changes that could be attributed to toxic or
even teratogenic effects. One case of intrauterine death was
reported (Reynoso and Huerta, 1994), which was probably
caused by the administration or dosage of idarubicin and
not by G-CSF; no similar cases were subsequently noted
with different chemotherapy protocols (Claahsen et al.,
1998).
With regard to PBSC or bone marrow harvesting during
pregnancy and G-CSF: healthy children, definitively free
from hematological abnormalities, were born to patients
with PBSC (4 days of 10 ␮g/kg/day as a donor for the
patient’s brother with nonHodgkin’s lymphoma (Leitner
et al., 2001), and 6 days at 16 ␮g/kg/day as consolidation
130
W. Würfel / Journal of Reproductive Immunology 108 (2015) 123–135
in acute myeloid leukemia (Niedermeier et al., 2005)) and
to the three bone marrow donors (Calder et al., 2005). A
recently published review (Pessach et al., 2013) concluded
that on the basis of currently available information, administration of G-CSF is safe even in pregnancy and that healthy
pregnant women can also donate PBSC and bone marrow
(after administration of G-CSF).
Table 5 presents available information on the course
of pregnancies and obstetrical outcomes of our RIF and
RSA patients (see also Würfel, 2014). This information is
based on voluntary disclosures, questionnaires, and telephone interviews (patients or treating gynecologists). The
data show no increased rate of malformations, preterm
births or genetically caused abortions. Genetic analysis of
abortion material was carried out where possible. Results
show that the majority of abortions under G-CSF are due
to genetic aberrations. It is also noteworthy that the number of induced abortions in the age group under treatment
(37–38 years) is relatively low – possibly an indication
that G-CSF promotes the abortion of genetically abnormal
embryos.
The SCNIR provides relatively extensive data on the
administration of G-CSF in pregnancy. The duration of
treatment of pregnant patients with chronic neutropenia
averaged two trimesters (minimum one, in some cases
three; average daily dose 2.7 ␮g/kg rh-G-CSF). No indications, or even proof, of higher morbidity or mortality in the
G-CSF group were observed, and the abortion rate tended to
be lower (Cottle et al., 2002; Dale et al., 2003). In 2010 Boxer
et al. – as already cited – reported on 60 pregnant patients
who received rh-G-CSF (average dose 1.07 ␮g/kg/day), and
observed a lower rate of abortions (5% versus 26% without
G-CSF), a higher rate of live births (88% versus 67% without
G-CSF), fewer severe maternal and neonatal complications,
and no significant increase in the rate of malformations. The
European branch of the SCNIR likewise noted no undesirable maternal or neonatal side-effects, and no teratogenic
effects in 21 pregnant women, 16 of whom were treated
with G-CSF throughout the entire term (Zeidler et al., 2014).
For this reason, women with chronic neutropenia are no
longer advised to avoid treatment with G-CSF in pregnancy.
7. Summary
Contrary to its name, G-CSF is a ubiquitous growth factor
involved in many control processes, and is also related to
reproduction. Based on the available studies, it influences
and promotes oocyte maturation, endometrial receptivity,
development of preimplantation embryos, and trophoblast
invasion. There is evidence that the pregnancy rate in
ART treatment increases, particularly in patients with RIF,
and that the abortion rate in patients with RSA decreases.
The small number of studies on these issues agree on all
these points. At the same time patents on issues such
as the reduction of preterm births or preeclampsia, have
been taken out. The application of G-CSF in reproductive
medicine or during pregnancy in the reported doses evidently does not imply a higher risk of maternal or neonatal
malignomas or of malformations in offspring. However, in
cases involving higher doses and longer duration of application, there is an increased risk of malignomas. Numerous
questions still await answers, such as what are the correct
indication, dosages, duration of administration, whether
any kind of ‘IGDS’ exists, and whether G-CSF is actually able
to reduce the risk of preeclampsia and/or preterm birth.
Conflict of interest
None declared.
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