J. Biol. Regul. Homeost. Agents.

2023; 37(4): 1933–1947

Article https://doi.org/10.23812/j.biol.regul.homeost.agents.20233704.192

Effect of Granulocyte Colony-Stimulating Factor on

Clinical Pregnancy Outcomes of Recurrent Miscarriage
and Recurrent Implantation Failure Cases: A
Meta-Analysis
Fangxiang Mu1 , Xin Yang1 , Mei Wang1 , Lin Liu1 , Fang Wang1, *
1 Department of Reproductive Medicine, Lanzhou University Second Hospital, 730000 Lanzhou, Gansu, China
*Correspondence: ery_fwang@lzu.edu.cn (Fang Wang)
Published: 20 April 2023

Objective: This meta-analysis aimed to evaluate the effect of granulocyte colony-stimulating factor (G-CSF) on the treatment of
recurrent miscarriage (RM) and recurrent implantation failure (RIF).
Methods: Eligible randomized controlled trials (RCTs) and cohort studies were retrieved from the Web of Science, Cochrane,
and PubMed databases and evaluated using the Jadad scale or Newcastle-Ottawa Scale. Cochran’s Q test and I2 statistics were
used to assess heterogeneity. The effect sizes for clinical pregnancy and abortion rates of patients were pooled as risk ratios (RRs)
and 95% confidence intervals (CI) using RevMan 5.3. Publication bias was assessed using funnel plots.
Results: Thirteen studies (nine RCTs and three cohort studies) involving 1262 participants were included. Compared to the
control/placebo group, the use of G-CSF significantly improved the clinical pregnancy rate [RRs (95% CI) = 1.73 (1.41, 2.12), p
< 0.00001] of RIF patients; Whereas it had no significant impact on their abortion rate [RRs (95% CI) = 1.13 (0.43, 2.95), p = 0.80].
Both subcutaneous and intrauterine injections of G-CSF could improve the clinical pregnancy rate in RIF patients. However,
subcutaneous injection showed a tendency to increase the abortion rate [RRs (95% CI) = 1.98 (0.40, 9.87), p = 0.40], whereas
intrauterine injection showed a tendency to decrease the abortion rate for RIF patients [RRs (95% CI) = 0.93 (0.24, 3.53), p =
0.11]. In addition, G-CSF use had no significant impact on the clinical pregnancy rate of RIF patients in a South American study
[RRs (95% CI) = 1.20 (0.60, 2.38), p = 0.60]. For RM patients, the use of G-CSF showed improved clinical pregnancy rates [RRs
(95% CI) = 1.43 (0.76, 2.70), p = 0.27] and lower abortion rates [RRs (95% CI) = 0.80 (0.46, 1.14), p = 0.44] than control/placebo
group; However, the difference was not significant. Similar results were observed in the subcutaneous, intrauterine injection,
and regions subgroups of RM patients.
Conclusions: This meta-analysis confirmed the benefits of G-CSF in improving the clinical pregnancy rate of RIF patients. No
conclusive evidence supports the link between G-CSF use and increased abortion rate in RIF patients and clarifies the association
of G-CSF use with clinical pregnancy and abortion rates in patients with RM.

Keywords: granulocyte colony-stimulating factor; recurrent miscarriage; recurrent implantation failure; clinical pregnancy rate; abortion
rate

Introduction a woman aged <40 years. Embryo implantation is a com-

Infertility is defined as a couple’s failure to achieve
pregnancy after one year or more of regular unprotected
sexual intercourse [1]. There is an estimated 8%–12% in-
fertility among reproductive-aged couples globally [2]. As-
sisted reproductive technology (ART) has emerged as a
common treatment for infertile patients; However, the rate
of pregnancy success and live birth remains unsatisfactory
[3], and recurrent implantation failure (RIF) is largely re-
sponsible for such ungratified outcomes.
Although there is no consensus on the definition of
RIF [4], Coughlan et al. [5] indicated that RIF refers
to pregnancy failure after transferring at least four good-
quality embryos in at least three fresh or frozen cycles in
plex process involving localization, adhesion, and invasion;
Its failure involves various aspects, including maternal age,
uterine factors (e.g., endometrial receptivity), embryo fac-
tors (e.g., competent blastocysts), and successful crosstalk
between the embryonic and maternal interfaces [6,7]. The
spontaneous loss of two or more consecutive pregnancies
before 20–24 weeks of gestation is considered a recurrent
miscarriage (RM), which is an early pregnancy complica-
tion affecting approximately 1–3% of couples trying to con-
ceive, also known as recurrent pregnancy loss [8,9]. RM
is another pathological entity sharing some of the causes
with RIF, including maternal endocrine, embryonic chro-
mosomal abnormalities, thrombophilia, and immunologi-

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1934
cal disturbances [10]. RIF and RM can occur separately
and simultaneously. Patients who had RIF and RM showed
particularly poor obstetric outcomes [11]. Immune system
imbalance is a cause of RIF and RM. For example, in-
creased cytotoxicity and levels of natural killer cells, in-
creased Th1:Th2 cell ratios, presence of various autoanti-
bodies, and dysregulated cytokines have been observed in
RIF and RM patients [12–16]. Although there are some
similarities in the pathogenesis and treatment of RM and
RIF, these two conditions represent different points of re-
productive failure, including infertility, RIF in women un-
dergoing in vitro fertilization (IVF), and RM [17].
Granulocyte colony-stimulating factor (G-CSF) is a
growth factor that has gained considerable attention in hu-
man reproduction following the initial case series of Gle-
icher et al. [18], who demonstrated the beneficial impact of
G-CSF in patients diagnosed with a thin endometrium. Sub-
sequently, G-CSF was found to play an important role in
improving ovarian function and embryo implantation, pro-
moting endometrial regeneration, and in the continuation of
pregnancy [19,20]. However, the benefits of G-CSF in r the
treatment of RM and RIF have not been sufficiently proven.
Several studies have demonstrated the application of G-
CSF in improving implantation and clinical pregnancy rates
[21,22]. In addition, studies have suggested that G-CSF has
no obvious effectiveness in improving clinical pregnancy
or live births [23] and may increase miscarriage rates [24].
Meta-analyses have reported positive effects of G-CSF in
improving the rates of implantation and clinical pregnancy
in RIF patients [25,26]. However, the association between
G-CSF use and miscarriage rate remains unclear. There-
fore, this study aimed to describe the effects of G-CSF in
clinical pregnancy and miscarriage rates in patients with
RIF and RM, based on a meta-analysis of randomized con-
trolled trials (RCTs) and cohort studies.
Materials and Methods
Strategies for Studies Retrieval
Relevant studies were searched from the Web of Sci-
ence, Cochrane, and PubMed databases following the pre-
defined search strategy by 14 July 2022, with no language
restrictions. The search words include medical subject
headings (“Granulocyte colony stimulating factor”, “Re-
current Miscarriage”, “Recurrent implantation failure”, and
“pregnancy Rate”) and their corresponding random word,
with the combination of subject words and free words used
for retrieval. Supplementary Table 1 lists the detailed
search strategies for each database. Additionally, the refer-
ences involved in the relevant reviews and included studies
were manually searched to obtain more eligible studies.
Screening Eligible Studies
Studies were selected if they met each of the follow-
ing criteria: (1) Studies that involved populations of pa-
tients with RM or RIF; (2) Interventions in the studies in-
cluded the use of G-CSF; (3) Clinical outcomes included
pregnancy rate and/or abortion rate; (4) Studies involved
efficacy comparison with placebo/control; And (5) random-
ized clinical trial (RCT) or cohort studies. Single-arm stud-
ies, non-original studies (reviews, letters, and comments),
and studies that contained combination drugs in the con-
trol group were excluded. Notably, only study with the
most complete information was included when the same
data were used in multiple studies, and the other studies
were excluded.
Data Extraction
Relevant data from the included studies were ex-
tracted, including the name of the first author, year of publi-
cation, country in which the research was conducted, study
design, characteristics of the participants (disease types,
numbers, age, number of previous failures, and interven-
tions), and clinical outcomes (frequency and rates of clin-
ical pregnancy and abortion). Data extraction was con-
ducted by two independent investigators, and any incon-
formity during this process was addressed through consul-
tation.
Quality Assessment
Cohort studies were assessed for selection, compara-
bility, exposure, and outcomes using the Newcastle-Ottawa
Scale (NOS) [27], which comprises a maximum score of 9.
Studies with scores >5 were considered to be of high qual-
ity. The methodological quality of RCT was assessed using
the Jadad scale, in which the study was assessed in terms
of whether it was randomized or double-blind, in addition
to whether withdrawals and dropouts were mentioned [28].
The Jadad scale has a maximum score of 7, and the study
was regarded as high quality with a score >4.
Statistical Analysis
RevMan5.3 software (The Cochrane Collaboration,
Oxford, UK) was utilized for all statistical analyses. The
effect sizes of categorical variables were pooled using risk
ratios (RRs) and 95% confidence intervals (CI). Hetero-
geneity among the studies was assessed using Cochran’s
Q and I2 tests. Significant heterogeneity existed with I2
>50% and/or p < 0.05, whereas no significant heterogene-
ity was found with I2 ≤50% and p > 0.05. Considering the
multiple influences of clinical trials from trial design, in-
tervention dose, regions of samples, age, and other factors,
a random-effects model was used to pool the effect size.
Subgroup analysis was conducted according to the mode of
administration, regions where studies were conducted, and
whether an adjusted analysis was performed to evaluate its
impact on heterogeneity and meta-analysis results. Publi-
cation bias was evaluated using funnel plots.

Results
Study Retrievalz
The study retrieval and selection process is shown in
Fig. 1. We selected 58 studies from three databases, and
four studies were manually selected. Among these 62 stud-
ies, 10 repetitive studies were excluded, and 25 studies that
met the inclusion criteria were excluded after reviewing the
titles and abstracts. After reading the full text, three studies
were excluded. Ultimately, 13 studies were included in the
analysis.
Fig. 1. Study selection process.
Characteristics of the Included Studies
There were 1262 participants involved in the 13 stud-
ies included in the analysis (Table 1, Ref. [21–23,29–38]).
These studies were conducted mainly in Iran and seven
other countries, including Germany, Hungary, and China.
Among the 13 studies, four involved RM, including three
RCTs [23,29,30] and one cohort study [22]. Nine studies
involved RIF, including seven RCTs [21,31–36] and two
cohort studies [37,38]. Additionally, two studies [33,34]
compared the clinical outcomes of RIF patients among the
G-CSF, placebo (saline), and control groups. The number
of previous failures of RIF patients was not mentioned in
a study by Kalem et al. [35], and the abortion rate of RIF
patients after interventions was not reported in three studies
[21,36,38]. Baseline factors were adjusted only in the study
by Aleyasin et al. [21].
The dosage, mode of administration, and duration of
G-CSF administration are listed in Table 2 (Ref. [21–
23,29–38]). G-CSF was administered by intrauterine or
subcutaneous injection; Both intrauterine and subcutaneous
injections were used in the study by Zeyneloglu et al. [38].
1935
The dosage and administration time differed among the
studies.
Among the 10 RCTs, three RCTs were assessed as
low quality due to the absence of a description of ran-
domized hiding, blind method, and/or withdraw and exit
(Supplementary Table 2). Quality assessment of the three
cohort studies categorized these studies into high-quality
studies with scores greater than 7, suggesting that there was
no significant study bias (Supplementary Table 3).
Association of G-CSF Use with Clinical Pregnancy
Rate
Four studies reported the clinical pregnancy rate of
RM patients, and significant heterogeneity was found
among the studies (I2 = 88%, p < 0.0001). The pooled
results indicated that the use of G-CSF tended to improve
the clinical pregnancy rate [RRs (95% CI) = 1.43 (0.76,
2.70), p = 0.27] of RM patients in comparison with that
of the control group; However, the difference was not sig-
nificant (Fig. 2A, Ref. [21–23,29–38]). Nine studies re-
ported the clinical pregnancy rates of RIF patients, and there
was no significant heterogeneity among the studies (I2 =
0%, p = 0.59). The pooled results indicated that the use of
G-CSF significantly improved the clinical pregnancy rate
[RRs (95% CI) = 1.73 (1.41, 2.12), p < 0.00001] of RIF pa-
tients in comparison with that of the control/placebo group
(Fig. 2A).
Association of G-CSF Use with Abortion Rate
There was significant heterogeneity (I2 = 57%, p =
0.07) among the four studies that reported the abortion rate
of RM patients. The pooled results revealed that the use of
G-CSF showed a trend toward lower abortion rates [RRs
(95% CI) = 0.80 (0.46, 1.14), p = 0.44] than the control
group, but the difference was not significant (Fig. 2B). Six
studies reported the abortion rate of RIF patients, and no
significant heterogeneity was observed among these stud-
ies (I2 = 30%, p = 0.21). Compared to the control/placebo
group, G-CSF use showed no significant impact [RRs (95%
CI) = 1.13 (0.43, 2.95), p = 0.80] on the abortion rate among
RIF patients (Fig. 2B).
Subgroups by Mode of G-CSF Administration
For RIF patients, subcutaneous [RRs (95% CI) = 1.61
(1.02, 2.53), p = 0.04] and intrauterine injection [RRs (95%
CI) = 1.72 (1.33, 2.22), p < 0.0001] of G-CSF improved
the clinical pregnancy rate (Fig. 3A, Ref. [21,28,31–33,35–
37]). In addition, no significant impact on abortion rate was
observed in the subcutaneous and intrauterine injection sub-
groups (Fig. 3B). However, subcutaneous injection showed
a tendency to increase the abortion rate [RRs (95% CI) =
1.98 (0.40, 9.87), p = 0.40], whereas intrauterine injection
showed a tendency to decrease the abortion rate among RIF
patients [RRs (95% CI) = 0.93 (0.24, 3.53), p = 0.11].
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Fig. 2. Effect of G-CSF on clinical pregnancy and abortion rates. Forest plots showing the pooled results of G-CSF use on clinical
pregnancy rate (A) and abortion rate (B) of recurrent miscarriage (RM) and recurrent implantation failure (RIF) patients.
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Fig. 3. Modes of G-CSF administration on clinical pregnancy and abortion rates among RIF patients. Forest plots showing
the pooled results of G-CSF subcutaneous or intrauterine injection on clinical pregnancy rate (A) and abortion rate (B) of recurrent
implantation failure (RIF) patients.
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Fig. 4. Modes of G-CSF administration on clinical pregnancy and abortion rates among RM patients. Forest plots showing
the pooled results of G-CSF subcutaneous or intrauterine injections on clinical pregnancy rate (A) and abortion rate (B) of recurrent
miscarriage (RM) patients.
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Fig. 5. Effect of G-CSF administration on clinical pregnancy and abortion rates among RM patients in different regions. Forest
plots showing the pooled results of G-CSF administration on clinical pregnancy rate (A) and abortion rate (B) of recurrent miscarriage
(RM) patients in Europe and West Asia.
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Fig. 6. Effect of G-CSF administration on clinical pregnancy and abortion rates for RIF patients in different regions. Forest plots
showing the pooled results of G-CSF administration on clinical pregnancy rate (A) and abortion rate (B) of recurrent implantation failure
(RIF) patients in West Asia, East Asia, South America, and Africa.
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Fig. 7. Subgroups by adjusted analysis. Forest plots showing the pooled results of G-CSF administration on clinical pregnancy rate
(A) and abortion rate (B) of recurrent implantation failure (RIF) patients in the adjusted or non-adjusted subgroups.
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Table 1. Characteristics of the included studies.
Author Country Study Diagnostic Participants Age (Y) Previous Clinical pregnancies Abortion rate Adjusted factors
(year) design system case vs control case/control failure
case/control
34.9 ± 2.7/
Scarpellini Hungary RCT RM 35 vs 33 ≥4/≥4 G-CSF: 29 (82.8%)/Placebo: 16 G-CSF: 6 (17.2%)/ Placebo: 17 None
33.8 ± 2.9
F. 2009 [29] (48.5%) (51.5%)
37.63 ± 3.97/
Santjohanser Germany cohort RM 49 vs 33 ≥2/≥2 G-CSF: 23 (46.9%)/control: 8 (24.4%) G-CSF: 7 (30.4%)/control: 4 (50%) None
37.61 ± 4.41
C. 2013 [22] study
age, endometrial thickness,
33.5 ± 4.2/ good-quality metaphase II oocyte counts,
Aleyasin A. Iran RCT RIF 56 vs 56 ≥3/≥3 G-CSF: 21 (37.5%)/control: 8 (14.3%) ——
32.4 ± 5.2 number of transferred embryos,
2016 [21]
and anti-Mullerian hormone levels
35.5 ± 4.32/
Davari- Iran RCT RIF 40 vs 40 vs 20 35.3 ± 3.98/ ≥3/≥3 G-CSF: 8 (80%)/Placebo: 4 (80%) G-CSF: 2 (20%)/Placebo: 1 None
Tanha F. 35.4 ± 4.01 /control: 2 (100%) (20%)/control: 0
2016 [33]
32.55 ± 4.61/
Eftekhar M. Iran RCT RIF 45 vs 45 ≥2/≥2 G-CSF: 13 (28.88%)/control: 6 (13.3%) —— None
31.75 ± 5.16
2016 [36]
30.2 ± 4/
Zafardoust Iran RCT RM 23 vs 27 ≥3/≥3 G-CSF: 4 (17.4%)/control: 3 (11.1%) G-CSF: 8 (33%)/control: 10 (37.5%) None
31.6 ± 5
S. 2017 [30]
34.53 ± 5.50/
Arefi S. Iran RCT RIF 32 vs 20 ≥2/≥2 G-CSF: 18 (56.2%)/control: 8 (40%) G-CSF: 2 (6.5%)/control: 1 (5%) None
34.05 ± 6.5
2018 [32]
37.8 ± 3.8/
Dieamant F. Brazil cohort RIF 33 vs 33 ≥2/≥2 G-CSF: 12 (36.4%)/control: 10 (30.3%) G-CSF: 3 (25.0%)/control: 1 (9.0%) None
37.8 ± 3.9
2019 [37] study
Eapen A. Britain RCT RM 76 vs 74 29∼34/26∼33 ≥3/≥3 rhG-CSF: 67 (88.2%)/Placebo: 69 rhG-CSF: 28 (36.8%)/Placebo: 25 None
2019 [23] (93.2%) (33.8%)
32.09 ± 4.21/
Huang P. China RCT RIF 52 vs 52 vs 59 32.07 ± 4.36/ ≥2/≥2 G-CSF: 28 (53.84%)/Placebo: 28 G-CSF: 2 (7.14%)/Placebo: 12 None
2020 [34] 32.40 ± 4.29 (53.84%)/control: 21 (35.60%) (42.86%)/control: 9 (42.85%)
34.61 ± 4.77/
Kalem Ziya. Korea RCT RIF 82 vs 75 —— G-CSF: 31 (37.8%)/Placebo: 20 G-CSF: 7 (8.5%)/Placebo: 2 (2.7%) None
34.92 ± 5.60
2020 [35] (26.7%)
32.8 ± 4.2/
Zeyneloglu Turkey cohort RIF 38 vs 34 ≥2/≥2 G-CSF: 20 (52.6%)/control: 8 (23.5%) —— None
34.2 ± 4.2
H. B. 2020 study
[38]
35.1 ± 5.04/
Torky H. Egypt RCT RIF 50 vs 50 ≥3/≥3 G-CSF: 28 (56%)/Placebo: 11 (22.9%) G-CSF: 1 (2%)/Placebo: 2 (4.2%) None
35.17 ± 4.23
2022 [31]
Note: RCT, randomized clinical trial; RIF, repetitive implantation failure; RM, recurrent miscarriage; G-CSF, granulocyte colony-stimulating factor; rhG-CSF, recombinant human granulocyte colony-
stimulating factor; hGM-CSF, human granulocyte/macrophage colony-stimulating factor.
 1943

For RM patients, subcutaneous injection [RRs (95%

CI) = 1.64 (0.71, 2.82), p = 0.33] of G-CSF showed a ten-
dency to improve the clinical pregnancy rate; However, but
difference was not significant (Fig. 4A, Ref. [22,23,29,30]).
Intrauterine injection of G-CSF in RM patients was reported
in one study, and it had no significant impact [RRs (95%
CI) = 1.57 (0.39, 6.28), p = 0.53] on the patient’s clinical
pregnancy rate (Fig. 4A). Subcutaneous [RRs (95% CI) =
0.75 (0.33, 1.70), p = 0.49] and intrauterine injections [RRs
(95% CI) = 0.94 (0.45, 1.98), p = 0.87] of G-CSF tended to
decrease the abortion rate; However, the difference was not
significant (Fig. 4B).

Subgroups by Regions and Adjusted Analysis

In Europe, G-CSF use tended to improve the clinical
pregnancy rate [RRs (95% CI) = 1.41 (0.71, 2.82), p = 0.33]
and decrease the abortion rate [RRs (95% CI) = 0.75 (0.33,
1.70), p = 0.49) among RM patients; However, the differ-
ence was not significant (Fig. 5, Ref. [22,23,29,30]). Simi-
lar results were observed in a study performed in West Asia
(Fig. 5).
For RIF patients, the results revealed that G-CSF use
could improve the clinical pregnancy rate among patients in
West Asia [RRs (95% CI) = 1.99 (1.42, 2.78), p < 0.0001],
East Asia [RRs (95% CI) = 1.47 (1.07, 2.01), p = 0.02],
and Africa [RRs (95% CI) = 2.55 (1.43, 4.53), p = 0.002].
One study performed in South America [RRs (95% CI) =
1.20 (0.60, 2.38), p = 0.60] showed that G-CSF use had no
significant impact on the clinical pregnancy rate of patients Fig. 8. Publication bias assessment. Funnel plots showing no
(Fig. 6A, Ref. [21,31–38]). Additionally, G-CSF use had significant publication bias for studies that reported the effect of
no significant impact on the abortion rate of patients (all p
G-CSF use on clinical pregnancy rate (A) and abortion rate (B).
> 0.05, Fig. 6B).
RM, recurrent miscarriage; RIF, recurrent implantation failure.
Additionally, baseline factors were adjusted in only
one study, and the results showed that G-CSF use could
improve the clinical pregnancy rate of RIF patients after Discussion
adjusting for baseline factors [RRs (95% CI) = 2.63 (1.27,
5.42), p = 0.009]. After removing this study, the pooled re-
In recent years, RM and RIF have deeply troubled cou-
sults of other studies showed similar findings (Fig. 7A, Ref.
ples of reproductive ages. Substantial efforts have been
[21,31–38]). Six studies reported the abortion rate among
made to improve the rate of clinical pregnancy and live
RIF patients, and adjusted analysis was not performed in
births in these patients. G-CSF is a specific hematopoietic
these studies. The pooled results suggested that G-CSF use
cytokine found in various tissues and cell types, including
had no significant impact on the abortion rate of RIF pa-
reproduction-related organs and cells [39]. For example,
tients (Fig. 7B).
G-CSF and its receptor have been demonstrated to express
in human placental cytotrophoblasts, syncytiotrophoblasts,
Publication Bias
decidual stromal cells, and endometrial gland cells, indicat-
Publication bias was evaluated using a funnel plot ing their important roles in decidual and placental functions
(Fig. 8), and the results showed there was no significant [40]. In vitro experiments suggested that G-CSF was in-
publication bias of the included studies for both clinical volved in regulating blastocyst formation of small follicles
pregnancy rate (p = 0.159 for RM and p = 0.260 for RIF) and the blastocyst stage after IVF [41] and improved em-
and abortion rate (p = 0.616 for RM and p = 0.639 for RIF). bryonic development capacity [42]. Furthermore, G-CSF
has been proposed to define oocyte developmental com-
petence in embryo implantation to predict IVF outcomes
[43,44]. Therefore, G-CSF has attracted increased attention
in the field of human reproduction. Increasing evidence has
demonstrated that G-CSF plays important roles in improv-
1944

Table 2. Intervention methods in each study.

Study Dosage Mode of Administration time
administration
RM
Scarpellini F. 2009 [29] 1 mg (100,000 U/Kgꞏd) SC injection From the sixth day after ovulation until the oc-
currence of menstruation or to the end of the
ninth week of gestation
Santjohanser C. 2013 [22] 11 patients 1 × 34 Mill IU/week and SC injection Starting at the day of embryo transfer (ET) un-
38 patients 2 × 13 Mill IU/week til the 12th week of pregnancy
Zafardoust S. 2017 [30] 300 µg IU injection The first was at the start of intervention and
twice in a cycle (the second was at 7 days af-
ter hCG (human chorionic gonadotropin) in-
jection)
Eapen A. 2019 [23] 130 µg SC injection At the same time each day for 9 weeks after
randomization
RIF
Aleyasin A. 2016 [21] 300 µg SC injection 1 h before transfer
Davari-Tanha F. 2016 [33] 300 µg IU infusion Simultaneously with transfer
Eftekhar M. 2016 [36] 300 µg IU infusion After ovarian puncture for transvaginal oocyte
retrieval
Arefi S. 2018 [32] 300 µg SC injection 30 min before transfer
Dieamant F. 2019 [37] 300 µg SC injection On the same day of PRP (platelet-rich plasma)
injection, and repeated weekly
Huang P. 2020 [34] 150 µg IU infusion On day 3 before the ET
Kalem Z. 2020 [35] 30 mIU/mL IU infusion Once a day on hCG day, before hCG injection
Zeyneloglu H B. 2020 [38] 48 mIU for IU, 100,000 IU/kg for SC IU and SC injection The IU route of G-CSF was employed on ovu-
lation triggering day. SC injection was started
on the day of oocyte retrieval and administered
for 15 days
Torky H. 2022 [31] 300 µg IU injection After oocyte retrieval
Note: IU, intrauterine; SC, subcutaneous.

ing embryo implantation and ovarian function, promoting
endometrial regeneration, and in the continuation of preg-
nancy [19,20].
Based on a multicenter RCT, Aleyasin et al. [21]
indicated that subcutaneous G-CSF administration before
implantation could significantly improve the implantation
(18% vs. 7.2%), clinical pregnancy (37.5% vs. 14.3%),
and chemical pregnancy rates (44.6% vs. 19.6%) for RIF
patients undergoing IVF; In addition, the associations were
also significant after adjustment for multiple possible in-
fluencing factors, including endometrial thickness, number
of good-quality oocytes and transferred embryos, age, and
anti-Mullerian hormone levels. Kalem et al. [35] demon-
strated that intrauterine infusion of G-CSF had no obvious
impact on endometrial thickness, pregnancy, and live birth
rates in RIF patients with a normal endometrium. Our cur-
rent meta-analysis suggests that G-CSF use significantly
improved the clinical pregnancy rate of RIF patients but did
not increase the abortion rate. Similar results have been re-
ported previously [25,26]. Although G-CSF showed po-
tential benefits in the improvement of clinical outcomes
in RIF patients, there is no consensus on a standard clini-
cal treatment (method of administration, dosage, and medi-
cation time). A meta-analysis demonstrated that subcuta-
neous or intrauterine infusion could improve the clinical
pregnancy rate among RIF patients [25]. Zeyneloglu et
al. [38] compared three routes of G-CSF administration
and found that intrauterine combined with subcutaneous
administration before ovulation resulted in better clinical
outcomes than intrauterine or subcutaneous administration
alone for RIF patients. It has been suggested that G-CSF re-
ceptors are found in various tissues and cell types, and the
systematic application of G-CSF might trigger more side ef-
fects in comparison with its local application. Additionally,
intrauterine administration of G-CSF might be easier and
safer for patients due to the direct effect on the endometrium
via this administration route.
For RM patients, the pooled results indicated that G-
CSF use showed a trend toward improved clinical preg-
nancy rates (RRs = 1.43) and lower abortion rates (RRs =
0.80); However, the difference was not significant. San-
tjohanser et al. [22] suggested that G-CSF application
for RM patients undergoing ART could improve the preg-
nancy (47% vs. 27%/24%) and live birth rates (32%
 1945

vs. 13%/14%) compared with that of the control/placebo Conclusions

groups, and ˂10% of patients experienced side effects.
However, Eapen et al. [23] indicated that administration In conclusion, compared with control/placebo, the use
of G-CSF to unexplained RM patients could not improve of G-CSF significantly improved the clinical pregnancy rate
the clinical pregnancy rates at 20 weeks or live birth rates of RIF patients and showed no evidence to increase the
(59.2% vs. 64.9%) compared to the placebo group. Re- abortion rate. For RM patients, although the use of G-CSF
portedly, G-CSF expression was reduced in the villi of showed a trend toward improved clinical pregnancy rates
RM patients, and G-CSF inhibition in the trophoblast could and lower abortion rates, the difference was not significant.
decrease the proliferation and migration of cells in vitro, Further studies based on more well-designed, high-quality
whereas intraperitoneal administration of G-CSF could de- RCTs with larger sample sizes are needed to clarify the ef-
crease the abortion rate in RM mice [45]. Scarpellini et fects of G-CSF in patients with RIF and RM.
al. [46] proposed that the clinical effectiveness of G-CSF
treatment for RM patients was achieved probably by medi- Availability of Data and Materials
ating the maternal immune response by recruiting decidual
All relevant data are contained in the manuscript and
Treg cells and facilitating trophoblast growth through the
its additional file.
increase of G-CSF and VEGF (vascular endothelial growth
factor) expression.
Author Contributions
This study had several limitations. (1) There is no con-
sensus on the definitions of RIF and RM [4,9]; Therefore, FM and XY—designed the study and drafted the
the definitions for RIF and RM differed among the included manuscript; MW and LL—made contribution to data ex-
studies. RIF was defined as at least two failed implantations traction and quality assessment; FM and MW—performed
in several studies [20,32,34,37] and at least three failed im- the statistical analysis and visualization; FW—contributed
plantations in the other studies; In addition, in each cycle, to design of the study and revised the manuscript. All au-
the number of high-quality embryos transferred varied from thors contributed to editorial changes in the manuscript. All
at least one to five among the studies. The differences in authors read and approved the final manuscript. All authors
terms of the definition of RM mainly focus on the num- have participated sufficiently in the work and agreed to be
ber of abortions, consecutive abortions, abortion gestational accountable for all aspects of the work.
week, and whether biochemical pregnancy was included.
The number of abortions in the included studies varied from Ethics Approval and Consent to Participate
at least two miscarriages to more than four miscarriages,
and abortion gestational weeks of RM patients was not men- Not applicable.
tioned in any of the studies. Patients with biochemical preg-
nancies were excluded from the study by Santjohanser et al. Acknowledgment
[22], whereas biochemical pregnancies were not mentioned
in other studies. RM was defined as three or more consecu- Not applicable.
tive or non-consecutive first-trimester losses in the study by
Eapen et al. [23]. However, at least two sequential unex- Funding
plained abortions or three non-sequential unexplained abor-
This study was supported by the Science Foundation
tions were reported in the study by Zafardoust et al. [30].
of Lanzhou University (Grant No. 071100132) and the Sci-
(2) There was no consensus on a standard clinical treatment
ence Foundation of Lanzhou University Second Hospital
(method of administration, dosage, and medication time),
(Grant No. YJS-BD-19).
and these factors differed among studies. We performed
a subgroup analysis based only on the method of admin-
Conflict of Interest
istration. These differences among the studies may have
affected the results. Considering the multiple influences of The authors declare no conflict of interest.
clinical trials from trial design, intervention dose, regions
of samples, age, and other factors, a random-effects model Supplementary Material
was used to pool the effect size in this meta-analysis. (3)
Only clinical pregnancy and abortion rates were analyzed, Supplementary material associated with this article
and other clinical outcomes (e.g., live birth rate) were not can be found, in the online version, at https://doi.org/10.
analyzed. (4) Among the 10 RCT studies, three RCTs were 23812/j.biol.regul.homeost.agents.20233704.192.
assessed as low quality, which might have affected the pub-
lication bias to some extent. Although the degree of disper-
sion was within the acceptable range, there was no signifi-
cant publication bias.
1946
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