Abstract
BACKGROUND
Treatment of thin endometrium with granular leukocyte-colony stimulating factor (G-CSF) remains controversial.
AIM
To investigate the effect of G-CSF on the outcome of frozen embryo transfer in patients with thin endometrium.
METHODS
A retrospective propensity score matching (PSM) study was performed to assess patients administered frozen embryo transfer at the Reproductive Medicine Center of the Affiliated Drum Tower Hospital of Nanjing University Medical School, in 2012-2018. The patients were divided into G-CSF intrauterine perfusion (G-CSF) and non-G-CSF groups, and clinical pregnancy, implantation, ectopic pregnancy, and early abortion rates between the two groups were compared.
RESULTS
Before PSM, 372 cycles were enrolled, including 242 and 130 cycles in the G-CSF and non-G-CSF groups, respectively. Age (34.23 ± 5.76 vs 32.99 ± 5.59 years; P = 0.047) and the blastula/cleavage stage embryo ratio (0.68 vs 0.37; P = 0.011) were significantly elevated in the G-CSF group compared with the non-G-CSF group; however, clinical pregnancy (46.28% vs 51.54%; P = 0.371) and embryo implantation (35.21% vs 35.65%; P = 0.910) rates were similar in both groups. After PSM by age and blastula/cleavage stage embryo ratio, 244 cycles were included (122 cases each in the G-CSF and non-G-CSF groups). The clinical pregnancy (50.82 % vs 48.36%; P = 0.701) and embryo implantation (37.38% vs 34.11%; P = 0.480) remained similar in both groups.
CONCLUSION
Intrauterine infusion of G-CSF does not improve the clinical outcome of frozen embryo transfer in patients with thin endometrium.
Keywords: Thin endometrium, Granular leukocyte-colony stimulating factor, Intrauterine perfusion, Frozen embryo transfer
Core Tip: Granular leukocyte-colony stimulating factor (G-CSF) administration for the treatment of thin endometrium remains controversial. A retrospective study of patients with thin endometrium who underwent frozen embryo transfer (FET) at the Reproductive Medicine Center of the Affiliated Drum Tower Hospital of Nanjing University Medical School, from January 1, 2012 to December 31, 2018, was performed. This study suggested that G-CSF intrauterine infusion does not increase clinical pregnancy and embryo implantation rates after FET in patients with thin endometrium. Early abortion may be somewhat decreased by G-CSF administration.
INTRODUCTION
Successful embryo implantation depends on two key factors, including a well-developed embryo and a well-receptive endometrium[1]. Endometrial receptivity refers to the ability of the endometrium to receive embryos, and encompasses endometrial thickness, morphology, and blood flow distribution[2]. Endometrial thickness is one of the most important factors predicting the outcome of pregnancy in assisted reproduction[3,4]. Studies have shown that women with a thin endometrium have more difficulty obtaining good pregnancy outcome compared with those with normal endometrium[5]. Indeed, an endometrium thinner than 7 mm results in decreased odds ratio of achieving pregnancy both in women with pathologic and idiopathic factors[6]. Clinically, approximately 2.4% of patients undergoing in vitro fertilization (IVF) show refractory thin endometrium with no response to treatment, and represents a challenging issue[4].
Various treatment options for thin endometrium have been proposed, including high estradiol doses, human chorionic gonadotropin, tamoxifen, pentoxifylline, vitamin E, L-arginine, aspirin, nitroglycerin patches, vaginal sildenafil, acupuncture and neuromuscular electric stimulation, intrauterine administration of granulocyte colony stimulating factor (G-CSF), and stem cell therapy[6-12]. However, G-CSF administration for the treatment of thin endometrium remains controversial. It was first applied by Gleicher and colleagues in patients with thin endometrium in 2011, with increased endometrial thickness and successful pregnancy[13]. However, other studies suggested that G-CSF does not improve pregnancy outcome in patients with thin endometrium[6,14,15].
The above reports clearly indicated that further investigation is required before the wide application of G-CSF in patients with thin endometrium. Therefore, the current study aimed to investigate the effect of G-CSF on the outcome of frozen embryo transfer in patients with thin endometrium, using a propensity score matching (PSM) design.
MATERIALS AND METHODS
Subjects
A retrospective study of patients with thin endometrium who underwent frozen embryo transfer (FET) at the Reproductive Medicine Center of the Affiliated Drum Tower Hospital of Nanjing University Medical School, from January 1, 2012 to December 31, 2018 was performed.
Inclusion criteria were: (1) At least one transplantation cycle with an endometrium thickness < 8 mm; (2) At least one high-quality embryo available for freezing; and (3) Endometrium prepared with estrogen replacement treatment. Exclusion criteria were: uterine malformations, intrauterine adhesions, adenomyosis, endometrial polyps, and endometrial tuberculosis.
This study was approved by the ethics committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School. The patients who received intrauterine infusion of G-CSF provided signed informed consent before treatment. For this retrospective study, the requirement for informed consent was waived.
Endometrial preparation protocol
Both groups of patients underwent estrogen and progesterone replacement therapy. From the third day of menstrual bleeding, oral Femoston (Solvay Pharmaceuticals, Netherlands) at 6-8 mg/d was administered, and endometrial thickness was observed at 18-20 d of medication by vaginal ultrasound (Voloson; GE, United States) with a RIC5-9W-RS intracavity probe. Patients with endometrium thickness below 8 mm were asked about their willingness to try the G-CSF perfusion treatment. Intrauterine infusion of G-CSF was therefore performed according to the patient's wishes (G-CSF group); the remaining patients formed the non-G-CSF group. Then, Femoston and progestin (6 mg/d, respectively) and progesterone injection (Zhejiang Xianyu Pharmaceutical Co., Ltd., China) at 60 mg/d (20 mg/branch) were administrated for endometrial transformation. On the fifth day of progestogen administration, intrauterine transplantation of cleavage stage embryos was performed; alternatively, intrauterine transplantation of blastulas was performed on the seventh day of progestogen administration.
Intrauterine perfusion of G-CSF
In the G-CSF group, 1 mL of recombinant human G-CSF (150 μg/branch, 0.5 mL, 0.9 × 109 IU; Jie Xin, Jiangsu Wuzhong Pharmaceutical Group and Suzhong Zhongkai Biopharmaceutical Company, China) were transvaginally administered into the uterine cavity through artificial insemination tubes (J-IUIC-351304; COOK, United States) on the day before progestin treatment.
The patient was placed in the lithotomy position on the transplantation bed. After scrubbing the vulva and placing a sterile drape and the speculum, the vagina and the cervical canal were gently scrubbed. An artificial insemination tube was placed into the uterine cavity, and withdrawn after drug administration. Transplantation surgeries were performed by the same team. All participating physicians had more than 2 years of work experience, with job titles of junior and above.
Embryo transplantation
On the day of transplantation, after observing bladder filling by abdominal ultrasound, the patient was placed in the lithotomy position on the transplantation bed. After scrubbing the vulva and placing a sterile drape and the speculum, the vagina and cervical canal were gently scrubbed. A transfer catheter was gently placed under the guidance of abdominal ultrasound, followed by embryo injection. The transfer catheter was then withdrawn, ensuring that the embryo was released. All transplantations were performed by the same team, and the participating physicians and embryologists had > 3 years of work experience, with titles of intermediate senior or senior. The clinical pregnancy rate of artificial insemination in this center was stable as assessed by week, month and year.
Criteria for high quality embryo
Embryos were graded according to the Istanbul Consensus (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011)[16]. High-quality embryos at the cleavage stage included Class I (translucent cells with equal size, with no particles in the cytoplasm and ≤ 5% fragments) and class II (cells with slightly uneven size, with particles in the cytoplasm and 6%-20% fragments) embryos after thawing. High quality blastulas were those with an expansion grade ≥ IV and the trophoblast and inner cell mass not containing C (IVAA, IVAB, IVBB, VAA, VAB and VBB).
Assessment of pregnancy outcome
The serum human chorionic gonadotropin (HCG) index was detected at 14 days after transplantation. HCG-positive patients were those with HCG amounts above the reference range for non-pregnant women (0-5 IU/mL in our center). Ultrasound examination was performed at 28 d after transplantation, and patients with a gestational sac were diagnosed with clinical pregnancy. Successful embryo implantation was reflected by a gestational sac (including intrauterine and ectopic pregnancy) observed by ultrasound. Each pregnancy sac was counted as an embryo. Gestational sacs located in the fallopian tube, pelvis, uterine horn, cervix, cesarean scar, and abdominal cavity indicated ectopic pregnancy. Termination of a pregnancy at less than 12 gestation weeks due to non-human factors was considered early spontaneous abortion.
Observation indicators
Demographic features, including age, BMI, infertility years, primary or secondary infertility, were collected from medical records. Observation outcomes included clinical pregnancy (clinical pregnancy cycles/transplantation cycles × 100%), embryo implantation (number of embryos implanted/total number of embryo transplanted × 100%), ectopic pregnancy (number of cycles with ectopic pregnancy/total number of clinical pregnancy cycles × 100%), and early abortion (number of early spontaneous abortion cycles/total number of clinical pregnancy cycles × 100%) rates.
Statistical analysis
Data analysis was performed with the SPSS 24.0 software (IBM Corp., Armonk, NY, United States). Continuous variables were presented as mean ± SD, and categorical data were shown as frequency and percentage. The propensity score matching method was used to match baseline data with 1: 1. The t-test was carried out for comparing continuous variables. The chi-square test was used for comparing categorical data; variables not meeting the criteria for the chi-square test were compared by the Fisher's exact test. P < 0.05 was considered statistically significant.
RESULTS
Baseline patient characteristics
A total of 372 cycles (in 348 patients) were assessed in this study, including 242 and 130 cases in the G-CSF and non-G-CSF groups, respectively. Age (34.23 ± 5.76 vs 32.99 ± 5.59 years; P = 0.047) and the blastula/cleavage stage embryo ratio (0.68 vs 0.37; P = 0.011) were significantly elevated in the G-CSF group compared with the non-G-CSF group. The remaining baseline characteristics were similar in both groups (Table 1).
Table 1.
Baseline patient features and pregnancy outcomes in the whole study population
|
Characteristics
|
G-CSF group (n = 242)
|
Non-G-CSF group (n = 130)
|
P
value
|
| Age (yr), mean ± SD | 34.23 ± 5.76 | 32.99 ± 5.59 | 0.047 |
| BMI (kg/cm2), mean ± SD | 21.35 ± 3.97 | 20.89 ± 2.63 | 0.182 |
| Average infertility years (yr), mean ± SD | 5.13 ± 1.75 | 4.92 ± 1.81 | 0.281 |
| Primary infertility cycle, n (%) | 79 (32.64) | 53 (40.77) | 0.118 |
| Secondary infertility cycle, n (%) | 163 (67.36) | 77 (59.23) | |
| Average endometrial thickness (mm), mean ± SD | 7.16 ± 0.40 | 7.18 ± 0.43 | 0.655 |
| Cycle with one transplanted embryo, n (%) | 71 (29.34) | 30 (23.08) | 0.195 |
| Cycle with two transplanted embryos, n (%) | 171 (70.66) | 100 (76.92) | |
| Cycle with blastula transplantation, n (%) | 97 (40.08) | 35 (26.92) | 0.011 |
| Cycle with cleavage stage embryo transplantation, n (%) | 145 (59.92) | 95 (73.08) | |
| Clinical pregnancy rate, % | 46.28 (112/242) | 51.54 (67/130) | 0.371 |
| Embryo implantation rate, % | 35.21 (144/409) | 35.65 (82/230) | 0.910 |
| Ectopic pregnancy rate, % | 2.68 (3/112) | 1.49 (1/67) | > 0.9991 |
| Early abortion rate, % | 14.29 (16/112) | 22.39 (15/67) | 0.166 |
Fisher’s exact test. G-CSF: Granular leukocyte-colony stimulating factor; BMI: Body mass index.
Pregnancy outcomes in the whole patient population
Comparing the 242 and 130 cases in the G-CSF and non-G-CSF groups, there were no statistically significant differences in clinical pregnancy (46.28% vs 51.54%; P = 0.371), embryo implantation (35.21% vs 35.65%; P = 0.910), ectopic pregnancy (2.68% vs 1.49%; P > 0.999) and early abortion (14.29% vs 22.39%; P = 0.166) rates (all P > 0.05; Table 1). There were no adverse reactions in either groups.
Pregnancy outcomes after PSM
As shown above, there were statistically significant differences in age and blastula/cleavage stage embryo ratio between the two groups in the whole patient population. Therefore, the propensity score matching method was used to match both groups of these parameters. As a result, 122 cases were included in each of the G-CSF and non-G-CSF groups. As shown in Table 2, there were no statistically significant differences in clinical pregnancy (50.82% vs 48.3%; P = 0.701), embryo implantation (37.38% vs 34.11%; P = 0.480), ectopic pregnancy (3.23% vs 1.69%; P = 0.965), and early abortion (12.90% vs 23.73%; P = 0.123) rates between the two groups.
Table 2.
Baseline patient features and pregnancy outcomes after propensity score matching based on age and blastula/cleavage stage embryo ratio
|
Characteristics
|
G-CSF group (n = 122)
|
Non-G-CSF group (n = 122)
|
P
value
|
| Age (yr), mean ± SD | 33.54 ± 5.97 | 33.45 ± 5.45 | 0.902 |
| BMI (kg/cm2), mean ± SD | 21.17 ± 2.33 | 20.79 ± 2.71 | 0.241 |
| Average infertility years (yr), mean ± SD | 4.77 ± 1.98 | 4.95 ± 1.93 | 0.473 |
| Primary infertility cycle, n (%) | 45 (32.64) | 50 (40.77) | 0.512 |
| Secondary infertility cycle, n (%) | 77 (67.36) | 72 (59.23) | |
| Average endometrial thickness (mm), mean ± SD | 7.13 ± 0.43 | 7.17 ± 0.43 | 0.468 |
| Cycle with one transplanted embryo, n (%) | 30 (24.59) | 30 (24.59) | > 0.999 |
| Cycle with two transplanted embryos, n (%) | 92 (75.41) | 92 (75.41) | |
| Cycle with blastula transplantation, n (%) | 35 (28.69) | 35 (28.69) | > 0.999 |
| Cycle with cleavage stage embryo transplantation, n (%) | 87 (71.31) | 87 (71.31) | |
| Clinical pregnancy rate, % | 50.82 (62/122) | 48.3 (59/122) | 0.701 |
| Embryo implantation rate, % | 37.38 (80/214) | 34.11 (73/214) | 0.480 |
| Ectopic pregnancy rate, % | 3.23 (2/62) | 1.69 (1/59) | 0.965 |
| Early abortion rate, % | 12.90 (8/62) | 23.73 (14/59) | 0.123 |
G-CSF: Granular leukocyte-colony stimulating factor; BMI: body mass index.
Pregnancy outcomes in cycles with blastula transplantation or cleavage stage embryo transplantation after PSM
Considering the possible effect of embryo transfer type on pregnancy outcome, the results were compared between blastula transplantation and cleavage stage embryo transplantation. Totally 35 blastula transplantation cases were included in each of the G-CSF and non-G-CSF groups. As shown in Table 3, there were no statistically significant differences in clinical pregnancy (68.57% vs 57.14%; P = 0.322), embryo implantation (57.41% vs 44.64%; P = 0.181), ectopic pregnancy (4.17% vs 0%; P > 0.999) and early abortion (20.83% vs 22.50%; P = 0.974) rates between the two groups. A total of 87 cases with cleavage stage embryo transplantation were included in each of the G-CSF and non-G-CSF groups. As shown in Table 4, there were no statistically significant differences in clinical pregnancy (43.68% vs 44.83%; P = 0.879), embryo implantation (30.63% vs 30.38%; P = 0.962), ectopic pregnancy (2.68% vs 01.49%; P > 0.999) and early abortion (14.29% vs 22.39%; P = 0.066) rates between the two groups.
Table 3.
Baseline patient features and pregnancy outcomes in cycles with blastula transplantation after propensity score matching based on age and blastula/cleavage stage embryo ratio
|
Characteristics (blastula)
|
G-CSF group (n = 35)
|
Non-G-CSF group (n = 35)
|
P
value
|
| Age (yr), mean ± SD | 31.26 ± 4.80 | 31.71 ± 4.46 | 0.686 |
| BMI (kg/cm2), mean ± SD | 21.93 ± 2.41 | 21.01 ± 2.65 | 0.133 |
| Average infertility years (yr), mean ± SD | 4.68 ± 1.91 | 4.80 ± 1.98 | 0.797 |
| Primary infertility cycle, n (%) | 13 (37.14) | 16 (45.71) | 0.467 |
| Secondary infertility cycle, n (%) | 22(72.86) | 19(54.29) | |
| Average endometrial thickness (mm), mean ± SD | 7.10 ± 0.53 | 7.07 ± 0.54 | 0.815 |
| Cycle with one transplanted embryo, n (%) | 16 (45.71) | 14 (40.00) | 0.233 |
| Cycle with two transplanted embryos, n (%) | 19 (54.29) | 21 (60.00) | |
| Clinical pregnancy rate, % | 68.57 (24/35) | 57.14 (20/35) | 0.322 |
| Embryo implantation rate, % | 57.41 (31/54) | 44.64 (25/56) | 0.181 |
| Ectopic pregnancy rate, % | 4.17 (1/24) | 0 (0/20) | > 0.9991 |
| Early abortion rate, % | 20.83 (5/24) | 22.50 (5/20) | 0.9742 |
Fisher’s exact test.
Corrective Chi-square test. G-CSF: Granular leukocyte-colony stimulating factor; BMI: body mass index.
Table 4.
Baseline patient features and pregnancy outcomes in cycles with cleavage stage embryo transplantation after propensity score matching based on age and blastula/cleavage stage embryo ratio
|
Characteristics (cleavage stage embryo )
|
G-CSF group (n = 87)
|
Non-G-CSF group (n = 87)
|
P
value
|
| Age (yr), mean ± SD | 35.29 ± 6.18 | 34.91 ± 5.67 | 0.673 |
| BMI (kg/cm2), mean ± SD | 20.86 ± 2.25 | 20.70 ± 2.73 | 0.674 |
| Average infertility years (yr), mean ± SD | 4.81 ± 2.13 | 5.01 ± 1.90 | 0.514 |
| Primary infertility cycle, n (%) | 32 (36.78) | 34 (39.08) | 0.755 |
| Secondary infertility cycle, n (%) | 55 (63.22) | 53 (60.92) | |
| Average endometrial thickness (mm), mean ± SD | 7.17 ± 0.39 | 7.26 ± 0.38 | 0.125 |
| Cycle with one transplanted embryo, n (%) | 14 (16.09) | 16 (18.39) | 0.161 |
| Cycle with two transplanted embryos, n (%) | 73 (83.91) | 71 (81.61) | |
| Clinical pregnancy rate, % | 43.68 (38/87) | 44.83 (39/87) | 0.879 |
| Embryo implantation rate, % | 30.63 (49/160) | 30.38 (48/158) | 0.962 |
| Ectopic pregnancy rate, % | 2.68 (1/38) | 1.49 (1/39) | > 0.9991 |
| Early abortion rate, % | 14.29 (3/38) | 22.39 (9/39) | 0.066 |
Fisher’s exact test. G-CSF: Granular leukocyte-colony stimulating factor; BMI: body mass index.
DISCUSSION
Reports have shown that CRL is larger in the first trimester in frozen-thawed embryo transfer compared with fresh embryo transfer[17]. Decreased risks of small for gestational age, low birth weight and preterm delivery have also been found in frozen-thawed embryo transfer[18] . In addition, uterine artery PI is lower in frozen-thawed embryo transfer compared with fresh embryo transplantation both in the first trimester and across the whole gestation[19]. After conception, when uterine artery resistance index does not decrease correspondingly, central arterial blood pressure increases, resulting in pressure vasospasm, in turn causing increased blood pressure, forming a vicious cycle[20]. However, a large number of retrospective studies have confirmed that frozen-thawed embryo transfer has a greater risk of pregnancy hypertension compared with fresh embryo transfer[21]. In addition, embryonic cryo-thawing does not seem to prevent preterm birth in IVF pregnancies[22]. Many scholars believe that frozen-thawed embryo transfer can reduce the occurrence of pregnancy-related complications. Frozen-thawed embryo transfer is often used as a supplement after failed fresh embryo transfer cycle, and the mother's state at conception is closer to that of natural conception. Moreover, frozen-thawed embryo transfer is less affected by other confounders than fresh embryo transfer, so it was examined in this study.
The present study demonstrated that intrauterine infusion of G-CSF does not improve the clinical outcome of frozen embryo transfer in patients with thin endometrium.
The FET technology reduces ovarian hyperstimulation syndrome (OHSS) occurrence[23] and the number of repetitive egg retrievals, improving embryo utilization and increasing the rate of cumulative pregnancy, as an important part of assisted reproductive technology (ART). Endometrial thickness represents one of the most important parameters for evaluating endometrial receptivity, and is closely related to the pregnancy outcome of assisted reproduction technology[2]. Currently, no uniform definition of thin endometrium is available. Generally, it is considered that an endometrium with a thickness below 7-8 mm cannot effectively support embryo implantation and maintain subsequent pregnancy. Therefore, the pregnancy rate of patients with thin endometrium is significantly reduced[3,24,25]. We consider an endometrial thickness < 8 mm to indicate a thin endometrium. In FET cycles, clinical pregnancy and live birth rates are significantly lower in patients with endometrial thickness ≤ 8 mm compared with the > 8 mm group[26]. Therefore, thin endometrium severely affects the success rate of IVF and imposes tremendous mental and economic burdens on patients.
Gleicher firstly infused G-CSF into the uterine cavity in 4 patients with thin endometrium, who all showed increased endometrial thickness and achieved clinical pregnancy[27]. This technique was repeated for 21 similar cases; although endometrial thickness was increased, only 19.1% of patients achieved clinical pregnancy[13]. However, Gleicher et al[27] used a self-control design, and the sample size was small. Since then, G-CSF has been assessed in multiple studies, with discrepant findings. For example, Sarvi et al[28] administered G-SCF in the superovulation cycle, and endometrial thickness and implantation rate were significantly improved, while Barad et al[29] reported opposite results. In FET, Gao et al[30] indicated that intrauterine perfusion of G-SCF achieves significant improvements in endometrial thickness and implantation rate, while Check et al[31] has a different conclusion. It should be noted that study protocols, perfusion times, and G-CSF doses were not uniform among the above studies.
To investigate whether intrauterine G-CSF infusion improves the clinical outcome of FET in patients with thin endometrium, this study retrospectively analyzed 372 FET cycles (in 348 patients). The results showed that clinical pregnancy, implantation, ectopic pregnancy and early abortion rates were similar in the G-CSF and non-G-CSF groups, corroborating previous reports[14,30]. Early abortion rate in the G-CSF group was 14.29% vs 22.39% for the non-G-CSF group, suggesting a trend of reduction after G-CSF treatment. In baseline data, age and blastula/cleavage stage embryo ratio showed statistically significant differences between the two groups, with higher values in the G-CSF groups. Age is an important factor affecting the clinical outcome of IVF. As age increases, clinical pregnancy and live birth rates show a downward trend[32]. Meanwhile, it is generally admitted that the implantation rate of blastulas is higher than that of cleavage stage embryos. In agreement, previous data in our center showed that the clinical pregnancy rate of cleavage stage embryos is significantly lower than that of blastulas[33]. Considering that age and blastula/cleavage stage embryo ratio differences may bias the clinical results, corrections were made by the propensity score matching method. After matching for age and blastula/cleavage embryo ratio in this study, pregnancy outcomes were not significantly different between the two groups. Similar results were obtained after subgroup analysis (in cycles with blastula transplantation or cleavage stage embryo transplantation). However, a 10.83% reduction in the early abortion rate was found in the G-CSF group (12.90%) compared with the non-G-CSF group (23.73%), suggesting that early abortion may be somewhat decreased by G-CSF administration. This could be explained by the fact that G-CSF can improve endometrial angiogenesis and effectively promote the regeneration of endometrial cells[34,35]. In addition, G-CSF is considered to be closely related to the menstrual cycle and pregnancy[36]. Our center has demonstrated that intrauterine perfusion of G-CSF significantly increases vascular endothelial growth factor (VEGF) amounts in human endometrial stromal cells by 2.25 times, suggesting that G-CSF may upregulate endometrial VEGF and increase blood flow (data submitted elsewhere). VEGF is a key factor in the regulation of endometrium vascular growth. It was shown that VEGF expression and microvessel density are significantly reduced in glandular epithelial cells of thin endometrium[35]. In addition, VEGF amounts in serum, villi and decidual tissues from patients with spontaneous abortion are lower than those of normal pregnant women[37,38]. The elevated trend of abortion rate in patients with thin endometrium may be related to reduced VEGF amounts. Therefore, we speculate that the slight decrease observed in abortion rate after intrauterine perfusion of G-CSF may be related to the above mechanism.
G-CSF, a cytokine that can stimulate the differentiation of bone marrow hematopoietic cells, was initially mainly used in the treatment of blood system diseases associated with leukopenia; recently, it was found to promote angiogenesis and immune regulation and inhibit inflammatory reactions with overt neurotrophic effect, and has been widely applied in nervous, cardiovascular and endocrine system diseases[39].The expression of G-CSF receptor was found in ovarian granulosa cells, the endometrium and the placenta. G-CSF plays an important role in pregnancy. It not only affects embryo implantation and ovarian function, but also promotes endometrial thickening, and can even be used as a remedy for embryo implantation failure[40]. One of the most important causes of implantation failure is the thin endometrium[41,42]. G-CSF can increase the recruitment of regulatory T cells and DC cells in the endometrium, affecting the remodeling of endometrial blood vessels, the regulation of the immune environment in the uterus, and the expression of key genes in the cell adhesion pathway during the process of transplantation[43]. In recent years, many studies have suggested that G-CSF, as a glycoprotein, improves endometrial thickness and facilitates embryo implantation[44,45]. A meta-analysis also showed that G-CSF administration has a beneficial role in clinical outcome after embryo transfer by both routes of local infusion and systematic administration, especially in cases with RIF[46].
G-CSF promotes embryo cleavage and blastocyst formation[47], controlling endometrial vascular remodeling, local immune modulation and cellular adhesion pathways, thereby playing an important role in embryo development and implantation. However, intrauterine surgery has a certain risk of infection, especially in case of infection by pathogenic microorganisms such as mold and bacteria in the vagina, and has a close relationship with the doctor's operation. Although there is still a lack of studies assessing the drug toxicity and teratogenicity of intrauterine G-CSF infusion, the possibility of serious adverse reactions cannot be completely ruled out. Therefore, whether this technique should be selected as a means to improve the clinical outcome should consider its advantages and disadvantages in the light of each patient's situation.
In the fresh embryo transplantation cycle, the endometrium is generally thicker due to stimulation by ovulation therapy, and thin endometrium is relatively rare. In addition, in the fresh cycle, patients with thin endometrium are often treated with cycle abandonment and frozen-thawed embryo transplantation. The data are insufficient. It is not clear whether the use of G-CSF is more beneficial to the thin endometrium of the fresh embryo transplantation cycle.
The current study had limitations. First, it was a retrospective trial, with inherent shortcomings. In addition, it was a single-center study, and selection bias could not be ruled out. Finally, the sample was relatively small. Therefore, large well-designed multi-center studies are warranted to comprehensively determine the effects of G-CSF on pregnancy outcome in patients with thin endometrium.
CONCLUSION
This study suggested that G-CSF intrauterine infusion does not increase clinical pregnancy and embryo implantation rates after FET in patients with thin endometrium.
ARTICLE HIGHLIGHTS
Research background
Endometrial thickness is one of the most important factors predicting the outcome of pregnancy. Women with a thin endometrium have difficulty obtaining good pregnancy outcome. Treatment of thin endometrium with granular leukocyte-colony stimulating factor (G-CSF) remains controversial.
Research motivation
G-CSF administration for the treatment of thin endometrium remains controversial.
Research objectives
This study aimed to investigate the effect of G-CSF on the outcome of frozen embryo transfer in patients with thin endometrium.
Research methods
A retrospective propensity score matching (PSM) study was performed to assess patients administered frozen embryo transfer at the Reproductive Medicine Center of the Affiliated Drum Tower Hospital of Nanjing University Medical School, in 2012-2018. The patients were divided into G-CSF intrauterine perfusion (G-CSF) and non-G-CSF groups, and clinical pregnancy, implantation, ectopic pregnancy, and early abortion rates between the two groups were compared.
Research results
After PSM by age and blastula/cleavage stage embryo ratio, 244 cycles were included (122 cases each in the G-CSF and non-G-CSF groups). The clinical pregnancy (50.82 % vs 48.36%; P = 0.701) and embryo implantation (37.38% vs 34.11%; P = 0.480) remained similar in both groups.
Research conclusions
This study suggested that G-CSF intrauterine infusion does not improve the clinical outcome of frozen embryo transfer in patients with thin endometrium.
Research perspectives
It provides some ideas for the G-CSF treatment of patients with thin endometrium.However, large well-designed multi-center studies are warranted to comprehensively determine the effects of G-CSF on pregnancy outcome in patients with thin endometrium.
ACKNOWLEDGEMENTS
We are grateful to all participants and the involved medical staff.
Footnotes
Institutional review board statement: This study was approved by the ethics committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School (Approval No. 2020-064-01).
Informed consent statement: Patients were not required to provide informed consent because the analysis used anonymous clinical data that were obtained after each patient agreed to treatment by written consent.
Conflict-of-interest statement: All authors declare that there are no any conflicts of interest.
Manuscript source: Unsolicited manuscript
Peer-review started: March 12, 2021
First decision: July 26, 2021
Article in press: September 14, 2021
Specialty type: Obstetrics and gynecology
Country/Territory of origin: China
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P-Reviewer: Cavoretto PI S-Editor: Ma YJ L-Editor: A P-Editor: Xing YX
Contributor Information
Ying-Chun Zhu, Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, Jiangsu Province, China.
Yan-Xin Sun, Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, Jiangsu Province, China.
Xiao-Yue Shen, Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, Jiangsu Province, China.
Yue Jiang, Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, Jiangsu Province, China.
Jing-Yu Liu, Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, Jiangsu Province, China. lianjing1216@126.com.
Data sharing statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
- 1.Fox C, Morin S, Jeong JW, Scott RT Jr, Lessey BA. Local and systemic factors and implantation: what is the evidence? Fertil Steril. 2016;105:873–884. doi: 10.1016/j.fertnstert.2016.02.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Bassil S. Changes in endometrial thickness, width, length and pattern in predicting pregnancy outcome during ovarian stimulation in in vitro fertilization. Ultrasound Obstet Gynecol. 2001;18:258–263. doi: 10.1046/j.1469-0705.2001.00502.x. [DOI] [PubMed] [Google Scholar]
- 3.Momeni M, Rahbar MH, Kovanci E. A meta-analysis of the relationship between endometrial thickness and outcome of in vitro fertilization cycles. J Hum Reprod Sci. 2011;4:130–137. doi: 10.4103/0974-1208.92287. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Kasius A, Smit JG, Torrance HL, Eijkemans MJ, Mol BW, Opmeer BC, Broekmans FJ. Endometrial thickness and pregnancy rates after IVF: a systematic review and meta-analysis. Hum Reprod Update. 2014;20:530–541. doi: 10.1093/humupd/dmu011. [DOI] [PubMed] [Google Scholar]
- 5.Zhang X, Chen CH, Confino E, Barnes R, Milad M, Kazer RR. Increased endometrial thickness is associated with improved treatment outcome for selected patients undergoing in vitro fertilization-embryo transfer. Fertil Steril. 2005;83:336–340. doi: 10.1016/j.fertnstert.2004.09.020. [DOI] [PubMed] [Google Scholar]
- 6.Miralaei S, Ashrafi M, Arabipoor A, Zolfaghari Z, Taghvaei S. The incidence rate of unresponsive thin endometrium in frozen embryo transfer cycles: A case-series of therapy with granulocyte colony stimulating factor. Int J Reprod Biomed. 2019;17:923–928. doi: 10.18502/ijrm.v17i12.5797. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Chen MJ, Yang JH, Peng FH, Chen SU, Ho HN, Yang YS. Extended estrogen administration for women with thin endometrium in frozen-thawed in-vitro fertilization programs. J Assist Reprod Genet. 2006;23:337–342. doi: 10.1007/s10815-006-9053-1. [DOI] [PubMed] [Google Scholar]
- 8.Urman B, Mercan R, Alatas C, Balaban B, Isiklar A, Nuhoglu A. Low-dose aspirin does not increase implantation rates in patients undergoing intracytoplasmic sperm injection: a prospective randomized study. J Assist Reprod Genet. 2000;17:586–590. doi: 10.1023/A:1026491426423. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Madani T, Ahmadi F, Jahangiri N, Bahmanabadi A, Bagheri Lankarani N. Does low-dose aspirin improve pregnancy rate in women undergoing frozen-thawed embryo transfer cycle? J Obstet Gynaecol Res. 2019;45:156–163. doi: 10.1111/jog.13802. [DOI] [PubMed] [Google Scholar]
- 10.Zinger M, Liu JH, Thomas MA. Successful use of vaginal sildenafil citrate in two infertility patients with Asherman's syndrome. J Womens Health (Larchmt) 2006;15:442–444. doi: 10.1089/jwh.2006.15.442. [DOI] [PubMed] [Google Scholar]
- 11.Check JH, Graziano V, Lee G, Nazari A, Choe JK, Dietterich C. Neither sildenafil nor vaginal estradiol improves endometrial thickness in women with thin endometria after taking oral estradiol in graduating dosages. Clin Exp Obstet Gynecol. 2004;31:99–102. [PubMed] [Google Scholar]
- 12.Senturk LM, Erel CT. Thin endometrium in assisted reproductive technology. Curr Opin Obstet Gynecol. 2008;20:221–228. doi: 10.1097/GCO.0b013e328302143c. [DOI] [PubMed] [Google Scholar]
- 13.Gleicher N, Kim A, Michaeli T, Lee HJ, Shohat-Tal A, Lazzaroni E, Barad DH. A pilot cohort study of granulocyte colony-stimulating factor in the treatment of unresponsive thin endometrium resistant to standard therapies. Hum Reprod. 2013;28:172–177. doi: 10.1093/humrep/des370. [DOI] [PubMed] [Google Scholar]
- 14.Lian R, Wang X, Lin R, Zeng H, Zeng Y, Liu S. Evaluation of granulocyte colony-stimulating factor on the treatment of thin endometrium during frozen-thawed embryo transfer cycles: a retrospective cohort study. Gynecol Endocrinol. 2020;36:370–374. doi: 10.1080/09513590.2019.1658187. [DOI] [PubMed] [Google Scholar]
- 15.Kunicki M, Łukaszuk K, Liss J, Skowrońska P, Szczyptańska J. Granulocyte colony stimulating factor treatment of resistant thin endometrium in women with frozen-thawed blastocyst transfer. Syst Biol Reprod Med. 2017;63:49–57. doi: 10.1080/19396368.2016.1251505. [DOI] [PubMed] [Google Scholar]
- 16.Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26:1270–1283. doi: 10.1093/humrep/der037. [DOI] [PubMed] [Google Scholar]
- 17.Cavoretto PI, Farina A, Girardelli S, Gaeta G, Spinillo S, Morano D, Amodeo S, Galdini A, Viganò P, Candiani M. Greater fetal crown-rump length growth with the use of in vitro fertilization or intracytoplasmic sperm injection conceptions after thawed versus fresh blastocyst transfers: secondary analysis of a prospective cohort study. Fertil Steril. 2021;116:147–156. doi: 10.1016/j.fertnstert.2020.11.035. [DOI] [PubMed] [Google Scholar]
- 18.Zhang J, Du M, Li Z, Wang L, Hu J, Zhao B, Feng Y, Chen X, Sun L. Fresh versus frozen embryo transfer for full-term singleton birth: a retrospective cohort study. J Ovarian Res. 2018;11:59. doi: 10.1186/s13048-018-0432-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Cavoretto PI, Farina A, Gaeta G, Sigismondi C, Spinillo S, Casiero D, Pozzoni M, Vigano P, Papaleo E, Candiani M. Uterine artery Doppler in singleton pregnancies conceived after in-vitro fertilization or intracytoplasmic sperm injection with fresh vs frozen blastocyst transfer: longitudinal cohort study. Ultrasound Obstet Gynecol. 2020;56:603–610. doi: 10.1002/uog.21969. [DOI] [PubMed] [Google Scholar]
- 20.Gopalakrishnan K, Mishra JS, Chinnathambi V, Vincent KL, Patrikeev I, Motamedi M, Saade GR, Hankins GD, Sathishkumar K. Elevated Testosterone Reduces Uterine Blood Flow, Spiral Artery Elongation, and Placental Oxygenation in Pregnant Rats. Hypertension. 2016;67:630–639. doi: 10.1161/HYPERTENSIONAHA.115.06946. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Maheshwari A, Pandey S, Amalraj Raja E, Shetty A, Hamilton M, Bhattacharya S. Is frozen embryo transfer better for mothers and babies? Hum Reprod Update. 2018;24:35–58. doi: 10.1093/humupd/dmx031. [DOI] [PubMed] [Google Scholar]
- 22.Cavoretto PI, Giorgione V, Sotiriadis A, Viganò P, Papaleo E, Galdini A, Gaeta G, Candiani M. IVF/ICSI treatment and the risk of iatrogenic preterm birth in singleton pregnancies: systematic review and meta-analysis of cohort studies. J Matern Fetal Neonatal Med. 2020:1–10. doi: 10.1080/14767058.2020.1771690. [DOI] [PubMed] [Google Scholar]
- 23.Hou XN, Wang JX, Wang Y, Sun HX. Comparison of pregnancy outcome between fresh embryo transfer and frozen embryo transfer in IVF/ICSI for patients with high risk of ovarian hyperstimulation syndrome. Shengzhi Yixue Zazhi . 2014;23:376–379. [Google Scholar]
- 24.Casper RF. It's time to pay attention to the endometrium. Fertil Steril. 2011;96:519–521. doi: 10.1016/j.fertnstert.2011.07.1096. [DOI] [PubMed] [Google Scholar]
- 25.Basir GS, O WS, So WW, Ng EH, Ho PC. Evaluation of cycle-to-cycle variation of endometrial responsiveness using transvaginal sonography in women undergoing assisted reproduction. Ultrasound Obstet Gynecol. 2002;19:484–489. doi: 10.1046/j.1469-0705.2002.00685.x. [DOI] [PubMed] [Google Scholar]
- 26.Bu Z, Wang K, Dai W, Sun Y. Endometrial thickness significantly affects clinical pregnancy and live birth rates in frozen-thawed embryo transfer cycles. Gynecol Endocrinol. 2016;32:524–528. doi: 10.3109/09513590.2015.1136616. [DOI] [PubMed] [Google Scholar]
- 27.Gleicher N, Vidali A, Barad DH. Successful treatment of unresponsive thin endometrium. Fertil Steril. 2011;95:2123.e13–2123.e17. doi: 10.1016/j.fertnstert.2011.01.143. [DOI] [PubMed] [Google Scholar]
- 28.Sarvi F, Arabahmadi M, Alleyassin A, Aghahosseini M, Ghasemi M. Effect of Increased Endometrial Thickness and Implantation Rate by Granulocyte Colony-Stimulating Factor on Unresponsive Thin Endometrium in Fresh In Vitro Fertilization Cycles: A Randomized Clinical Trial. Obstet Gynecol Int. 2017;2017:3596079. doi: 10.1155/2017/3596079. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Barad DH, Yu Y, Kushnir VA, Shohat-Tal A, Lazzaroni E, Lee HJ, Gleicher N. A randomized clinical trial of endometrial perfusion with granulocyte colony-stimulating factor in in vitro fertilization cycles: impact on endometrial thickness and clinical pregnancy rates. Fertil Steril. 2014;101:710–715. doi: 10.1016/j.fertnstert.2013.12.016. [DOI] [PubMed] [Google Scholar]
- 30.Gao MX, Chen ST, Li LF, Zhao LH, Shi Q, Jia XL, Ma XL, Zhang XH. Granulocyte Colony-stimulating Factor Uterine Perfusion for Inadequate Endometrium. Shengzhi Yu Biyun. 2014;34:527–533. [Google Scholar]
- 31.Check JH, Choe JK, Summers-Chase D. Failure to increase the thickness of thin endometria with intrauterine infusion of granulocyte colony stimulating factor (G-CSF) Clin Exp Obstet Gynecol. 2016;43:332–333. [PubMed] [Google Scholar]
- 32.McPherson NO, Zander-Fox D, Vincent AD, Lane M. Combined advanced parental age has an additive negative effect on live birth rates-data from 4057 first IVF/ICSI cycles. J Assist Reprod Genet. 2018;35:279–287. doi: 10.1007/s10815-017-1054-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Ni XB, Wang SS, Zhang NY, Sun HX. Clinical outcomes of cleavage-stage embryo transfer vs blastocyst transfer in patients with high-risk of hyperstimulation syndrome. Shengzhi Yixue Zazhi . 2016;25:610–613. [Google Scholar]
- 34.Geller RB. Use of cytokines in the treatment of acute myelocytic leukemia: a critical review. J Clin Oncol. 1996;14:1371–1382. doi: 10.1200/JCO.1996.14.4.1371. [DOI] [PubMed] [Google Scholar]
- 35.Zhao J, Tian T, Zhang Q, Wang Y, Li Y. Use of granulocyte colony-stimulating factor for the treatment of thin endometrium in experimental rats. PLoS One. 2013;8:e82375. doi: 10.1371/journal.pone.0082375. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
- 36.Makinoda S, Mikuni M, Furuta I, Okuyama K, Sagawa T, Fujimoto S. Serum concentration of endogenous G-CSF in women during the menstrual cycle and pregnancy. Eur J Clin Invest. 1995;25:877–879. doi: 10.1111/j.1365-2362.1995.tb01699.x. [DOI] [PubMed] [Google Scholar]
- 37.Bagheri A, Kumar P, Kamath A, Rao P. Association of angiogenic cytokines (VEGF-A and VEGF-C) and clinical characteristic in women with unexplained recurrent miscarriage. Bratisl Lek Listy. 2017;118:258–264. doi: 10.4149/BLL_2017_051. [DOI] [PubMed] [Google Scholar]
- 38.Zhi Z, Yang W, Liu L, Jiang X, Pang L. Early missed abortion is associated with villous angiogenesis via the HIF-1α/VEGF signaling pathway. Arch Gynecol Obstet. 2018;298:537–543. doi: 10.1007/s00404-018-4802-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Chen CC, Chen RF, Wang YC, Li YT, Chuang JH, Kuo YR. Combination of a CD26 Inhibitor, G-CSF, and Short-term Immunosuppressants Modulates Allotransplant Survival and Immunoregulation in a Rodent Hindlimb Allotransplant Model. Transplantation. 2021;105:1250–1260. doi: 10.1097/TP.0000000000003504. [DOI] [PubMed] [Google Scholar]
- 40.Eftekhar M, Miraj S, Farid Mojtahedi M, Neghab N. Efficacy of Intrauterine infusion of granulocyte colony stimulating factor on patients with history of implantation failure: A randomized control trial. Int J Reprod Biomed. 2016;14:687–690. [PMC free article] [PubMed] [Google Scholar]
- 41.Asante A, Coddington CC, Schenck L, Stewart EA. Thin endometrial stripe does not affect likelihood of achieving pregnancy in clomiphene citrate/intrauterine insemination cycles. Fertil Steril. 2013;100:1610–4.e1. doi: 10.1016/j.fertnstert.2013.08.035. [DOI] [PubMed] [Google Scholar]
- 42.Xia L, Meng Q, Xi J, Han Q, Cheng J, Shen J, Xia Y, Shi L. The synergistic effect of electroacupuncture and bone mesenchymal stem cell transplantation on repairing thin endometrial injury in rats. Stem Cell Res Ther. 2019;10:244. doi: 10.1186/s13287-019-1326-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Kamath MS, Chittawar PB, Kirubakaran R, Mascarenhas M. Use of granulocyte-colony stimulating factor in assisted reproductive technology: A systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2017;214:16–24. doi: 10.1016/j.ejogrb.2017.04.022. [DOI] [PubMed] [Google Scholar]
- 44.Xie Y, Zhang T, Tian Z, Zhang J, Wang W, Zhang H, Zeng Y, Ou J, Yang Y. Efficacy of intrauterine perfusion of granulocyte colony-stimulating factor (G-CSF) for Infertile women with thin endometrium: A systematic review and meta-analysis. Am J Reprod Immunol. 2017;78 doi: 10.1111/aji.12701. [DOI] [PubMed] [Google Scholar]
- 45.Shi H, Lan L, Lv X, Sun L. Effect of pidotimod combined with azithromycin on children with mycoplasma pneumonia and the expression levels of IL-10 and G-CSF in serum. Exp Ther Med. 2019;18:1800–1806. doi: 10.3892/etm.2019.7725. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Zhang L, Xu WH, Fu XH, Huang QX, Guo XY, Zhang L, Li SS, Zhu J, Shu J. Therapeutic role of granulocyte colony-stimulating factor (G-CSF) for infertile women under in vitro fertilization and embryo transfer (IVF-ET) treatment: a meta-analysis. Arch Gynecol Obstet. 2018;298:861–871. doi: 10.1007/s00404-018-4892-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Cai L, Jeon Y, Yoon JD, Hwang SU, Kim E, Park KM, Kim KJ, Jin MH, Lee E, Kim H, Jeung EB, Hyun SH. The effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro maturation of porcine oocyte on subsequent embryonic development. Theriogenology. 2015;84:1075–1087. doi: 10.1016/j.theriogenology.2015.06.008. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.