ABSTRACT
The aim of the study was to assess the granulocyte-colony stimulating factor (G-CSF) effect on unresponsive thin (<7 mm) endometrium in women undergoing frozen-thawed embryo transfer at the blastocyst stage. A total of 62 women with thin unresponsive endometrium were included in the study, of which, 29 received a G-CSF infusion and 33 who opted out of the study served as controls. Patients in both groups had similar endometrial thickness at the time of the initial evaluation: 6.50 mm (5.50-6.80) in the G-CSF and 6.40 mm (5.50-7.0) in the control group. However, after the infusion endometrial thickness increased significantly in the G-CSF group in comparison with the controls (p=0.01), (Δ) 0.5 (0.02-1.2) (p=0.005). In the G-CSF group endometrium expanded to 7.90 mm (6.58-8.70) while in the control group to 6.90 mm (6.0-7.75). Five women in each group conceived. The clinical pregnancy rate was 5/29 (17.24%) in the G-CSF treated group and 5/33 (15.15%) in the control group (p>0.05). The live birth rate was 2/29 (6.89%) in the G-CSF group and 2/33 (6.06%) in the control group (p>0.05). We concluded that G-CSF infusion leads to an improvement in endometrium thickness but not to any improvement in the clinical pregnancy and live birth rates. Until more data is available G-CSF treatment should be considered to be of limited value in increasing pregnancy rate. Abbreviations: G-CSF: granulocyte colony-stimulating factor; M-CSF: macrophagecolony-stimulating factor; GM-CSF: granulocyte-macrophage colony-stimulating factor; FET: frozen embryo transfer; IVF: in vitro fertilization
Introduction
Molecular and immunological mechanisms in the endometrium play a crucial role in the implantation process [Sharkey Citation1998]. Growth factors, hormones, and cytokines produced by decidual cells are also involved in implantation [Psychoyos Citation1986]. Psychoyos described this complex mechanism in 1986 in his “Uterine Receptivity for Nidation” study. However, the clinical problem of women with unresponsive thin endometrium remains unresolved.
On one hand, clinical practice has shown that pregnancy occurs when the endometrium reaches more than 7 and preferably 9 mm [Al-Ghamdi et al. Citation2008; Isaacs et al. Citation1996; Weckstein et al. Citation1997]. It is 0.6-0.8% of patients who fail to reach that thickness [Weckstein et al. Citation1997]. On the other hand, the role of endometrial thickness on implantation may be considered questionable. Women with thin endometrium are usually older [Kasius et al. Citation2014] and the association between thin endometrium and worse reproductive outcomes may be related to a poor ovarian response [Coelho Neto et al. Citation2015]. Moreover women with endometrial thickness <6 mm have similar reproductive outcomes when using donor eggs [Dain et al. Citation2013]. Several authors proposed numerous therapies, including low-dose aspirin, sildenafil citrate, tocopherol, and pentoxifylline [Lédée-Bataille et al. Citation2002; Sher and Fisch Citation2002; Weckstein et al. Citation1997].
Colony stimulating factors include macrophage colony-stimulating factor (M-CSF) also known as colony stimulating factor 1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) also known as colony stimulating factor 2 (CSF-2), granulocyte-colony stimulating factor (G-CSF) also known as colony stimulating factor 3 (CSF-3). Doctors have been using recombinant G-CSF in immunology for various indications for over 20 years. It promotes type 2 T helper cell (Th2) cytokine secretion, recruitment of dendritic and T regulatory cells, and has a pro-angiogenic effect [Barash et al. Citation2003; Rutella et al. Citation2005]. According to preclinical studies G-CSF affects decidual macrophages, stimulates neutrophilic granulocytes proliferation and differentiation, and also influences implantation [Barash et al. Citation2003; Loke et al. Citation1995]. G-CSF is considered to be a factor preventing repeated miscarriages and implantation failures [Rahmati et al. Citation2014; Santjohanser et al. Citation2013; Scarpellini and Sbracia Citation2009; Wurfel et al. Citation2010]. Additionally G-CSF has no effect on embryonic chromosomal constitution [Agerholm et al. Citation2010; Loureiro et al. Citation2009].
Gleicher et al. evaluated the usefulness of G-CSF in endometrium expansion in women who previously had cancelled cycles due to unresponsive endometrium [Gleicher et al. Citation2011; Gleicher et al. Citation2013]. The results were very promising and indicated that G-CSF infusion could lead to an increase in endometrial thickness. In this study there was no record of any serious drug-related adverse effects of G-CSF. To date, there have been only a few studies on G-CSF infusion and its effect on endometrium thickness and clinical pregnancy in women who have undergone hormone replacement frozen-thawed blastocyst stage embryo transfer [Li et al. Citation2014; Xu et al. Citation2015]. In two recent studies Li et al. and Xu et al. presented higher embryo implantation and clinical pregnancy rates in the G-CSF groups who had frozen embryo transfer (FET) [Li et al. Citation2014; Xu et al. Citation2015]. The aim of our study was to investigate the G-CSF effects on unresponsive thin (<7 mm) endometrium in women undergoing FET at the blastocyst stage to add to the available research and bring us closer to reaching conclusions regarding the usefulness G-CSF in treating patients with unresponsive endometrium.
Results
A total of 62 patients, among 134 patients with previously cancelled in vitro fertilization (IVF) cycles due to thin unresponsive endometrium (<7 mm) undergoing IVF in a frozen cycle, fulfilled all inclusion criteria and were included in this study. Among the 62 participants, 29 women were treated with G-CSF (treatment group), and 33 (no G-CSF treatment) were defined as the control group. Patients in both groups were very similar (their baseline characteristics are shown in –). Patients in both groups had similar endometrial thickness at the time of the initial evaluation: 6.50 mm (5.50-6.80) in the G-CSF group and 6.40 mm (5.50-7.0) in the control group. However, after the G-CSF infusion the endometrial thickness increased significantly in the treatment group in comparison with the controls (p=0.01), (Δ) 0.5 (0.02-1.2) (p=0.005). In the G-CSF group endometrium expanded to 7.90 mm (6.58-8.70) while 6.90 mm (6.0-7.75) in the control group. Data concerning measurements and observed changes for both groups are shown in .
Table 1. Baseline patient characteristics and IVF cycle characteristics in women with thin endometrium treated with GCSF and control group (p < 0.05).
Table 2. Baseline patient characteristics and IVF cycle characteristics in women who conceived with thin endometrium treated with GCSF and control group (p < 0.05).
Table 3. Baseline patient characteristics and IVF cycle characteristics in women who did not conceive with thin endometrium treated with GCSF and control group (p < 0.05).
Table 4. Endometrial thickness in women before and after infusion of G-CSF who had frozen-thawed embryo transfer of blastocysts (FBT) and from control group (p < 0.05).
Five women conceived in each group. The clinical pregnancy rate was 5/29 (17.24%) in the G-CSF treated group and 5/33 (15.15%) in the control group. The live birth rate was 2/29 (6.89%) and 2/33 (6.06%), respectively. It is interesting to note that one of the patients who conceived in the G-CSF treated group had endometrium below 7 mm and two who conceived in the control group also had endometrium below 7 mm.
Discussion
We evaluated the effectiveness of G-CSF treatment prior to FET. The endometrium increased significantly for the 29 women in the treatment group. However, clinical pregnancy rates (G-CSF: 17.24% and control: 15.15%) and live birth rates (6.89% and 6.06%, respectively) were similar in both groups.
The first limitation of the study is a relatively small number of patients. It may be especially visible during the comparative analysis of patients who conceived with some of the not statistically significant results being false negatives (Type II error). The second limitation is that endometrial thickness is prone to detection bias. The measurements were subjective and the observed differences were relatively small. Data from the literature showed that differences up to 2 mm might occur even when the measurement is repeated on the same day [Martins et al. Citation2011].
Success of an IVF procedure depends on many factors including adequate thickness of endometrium. An endometrial thickness of over 7-9 mm is considered favorable [Al-Ghamdi et al. Citation2008; Weissman et al. Citation1999], although some data suggests that even 5-8 mm is inadequate [Shufaro et al. Citation2008]. Conversely, other studies claim no correlation between endometrium thickness and IVF outcome. An inadequate endometrium affects <1% of women undergoing IVF treatment [Al-Ghamdi et al. Citation2008]. These patients are unable to proceed with embryo transfer and need to have their embryos frozen. Various experimental treatments can be proposed to improve endometrial thickness in the subsequent cycles [Gleicher et al. Citation2013].
Gleicher et al. were the first to show the promising role of G-CSF on endometrium expansion in women with unresponsive endometrium [Gleicher et al. Citation2011]. Their case report described four patients who conceived following the G-CSF infusion. Tanaka et al. [Citation2000] concluded that G-CSF, acting in a autocrine and paracrine way, improves cAMP–mediated decidualization of human endometrial stromal cells. Additionally Pala et al. [Citation2014] showed that, in rats with induced diabetes mellitus, G-CSF could have antiapoptotic effects on the endometrium and it could also, potentially, be promising in preventing injury of endometrium in humans.
Another pilot cohort study evaluated IVF outcomes after G-CSF application in 21 patients with thin endometrium. Authors observed a 19.1% ongoing clinical pregnancy rate and concluded that G-CSF is useful in the treatment of chronically thin endometrium [Gleicher et al. Citation2013]. A case study by Lucena and Moreno-Ortiz [Citation2013] reported an increase in endometrial thickness after infusing 300 ug of G-CSF at the time of oocyte retrieval. Similar results were observed in a nonrandomized clinical trial conducted by Tehraninejad et al. [Citation2015] on a group of fifteen infertile women undergoing fresh IVF cycles (infusion of 300 ug/1 mL of G-CSF at the day of oocyte retrieval).
Kunicki et al. assessed thirty-seven patients with thin endometrium who underwent fresh IVF cycles. All patients had a history of thin unresponsive endometrium in previous cycles. Endometrial thickness increased 72 hours after infusion of 300 ug/1 mL of G-CSF and the clinical pregnancy rate was 18.9% [Kunicki et al. Citation2014]. Barad et al. showed that in normal IVF patients infusion of G-CSF did not affect endometrial thickness, implantation rates, nor clinical pregnancy rates. Still he left the final assessment on how G-CSF’s effects pregnancy open suggesting the need for more studies involving younger participants [Barad et al. Citation2014]. Check et al. [Citation2014] achieved similar results, however, their study was limited by a small group of patients.
Few studies evaluated the use of G-CSF in conjunction with FET and the results remain inconclusive. Li et al. [Citation2014], in a prospective study with 34 patients (40 cycles), presented no significant differences in implantation and clinical pregnancy rates between patients treated with G-CSF and controls. It is worth noting that even though the difference was not statistically significant the clinical pregnancy rate was higher in the G-CSF group than in the self-controlled group (30.30% vs. 20.00%) [Li et al. Citation2014]. Xu et al. found that endometrial thickness increased significantly in the G-CSF group. The clinical pregnancy rate was very high at 48.1% and significantly greater than in the control group [Xu et al. Citation2015].
Eftekha et al. in a non-randomized intervention clinical trial with 68 participants (34 treated with G-CSF, and 34 in control group) demonstrated that G-CSF did not improve endometrial thickness in infertile women with thin endometrium in FET cycles. Chemical (39.30% vs. 14.30%) and clinical pregnancy rate (32.10 % vs. 12%) was higher in the treated group than in the control but not statistically significant [Eftekhar et al. Citation2014]. Cohort studies that have investigated the effect of intrauterine perfusion of GCSF on endometrium and pregnancy outcomes in FET cycles are presented in .
Table 5. Previous cohort studies that have investigated the effect of intrauterine perfusion of granulocyte colony-stimulating factor on endometrium and pregnancy outcomes in frozen embryo transfer cycles.
Discrepancies between our results and other studies could be explained by different factors. First of all, different intervals between G-CSF infusion and first reassessment of the endometrium could play a role. For example in Gleicher et al. [Citation2013] reassessment was at 48 hours, in the study presented here and in Li et al. [Citation2014] and Xu et al. [Citation2015] reassessment was at 72 hours. These observations raise the issue about the appropriate interval between infusion and second reassessment of endometrium. We should also ask how many times should G-CSF be applied. In the Gleicher’s [Citation2013] study some patients had two G-CSF infusions. In the study presented here as in Li et al. [Citation2014] and Xu et al. [Citation2015] only one infusion was performed.
Another issue is the method of delivering G-CSF. In the study presented in the above and aforementioned trials of others, G-CSF was infused directly into the uterine cavity. There are studies in which G-CSF is given subcutaneously [Scarpellini and Sbracia Citation2009]. It should be, however, noted that in the cited study G-CSF was used to treat women with repeated pregnancy loss and not thin and unresponsive endometrium. It has not been studied if subcutaneous infusion could be more or less effective in the treatment of thin endometrium. No final conclusions have been drawn regarding which delivery system is better.
There is also a possibility of a synergistic effect of low-dose aspirin with G-CSF added to the protocol. Aspirin supplementation was routinely applied in our study. However, we do not think that it could have had a great impact on the results because all participants had previously canceled cycles due to thin endometrium despite the application of either low dose aspirin or other medications. It is also interesting that in the control group, following extended estrogen treatment, seventeen women failed to reach endometrium thickness of over 7 mm, but sixteen did have endometrium over 7 mm. In the G-CSF group only a single woman had endometrium below 7 mm after the infusion. We can speculate that in the control group it was the longer duration of estrogen treatment that resulted in the endometrium growth even without a G-CSF infusion.
One should take into consideration that there are opposing results with aspirin in unselected IVF patients on the endometrium thickness and pregnancy rates [Rubinstein et al. Citation1999; Urman et al. Citation2000; Weckstein et al. Citation1997]. For example in the study by Urman et al. low dose aspirin treatment did not increase implantation rate and endometrial thickness in women undergoing IVF [Urman et al. Citation2000]. In the study reported in this article, all the women had an increasing dosage of estradiol supplementation. Thus the increase in the thickness of the endometrium in the control group could result from the increased dosage of estradiol supplementation, synergistic effect of estradiol with aspirin, or other factors. Women receiving G-CSF underwent similar estradiol and aspirin treatment and we think that the G-CSF was the factor leading to the higher increase of the endometrium.
In general, when discussing thin endometrium related issues, we should point out that the threshold of the so called ‘thin endometrium’ varies in different studies. Clinical pregnancy was observed even in women with endometrium <4 mm [Rubinstein et al. Citation1999]. Kaius et al. [Citation2014] argued that current data indicated that endometrial thickness had a limited capacity to identify women who have a low chance to conceive after IVF.
Another procedure, which is said to help increase endometrial receptivity, is ‘endometrium scratching’ prior to the embryo transfer and some authors show that it improves implantation rates [Tiboni et al. Citation2011; Zhou et al. Citation2008]. This procedure was not performed in our study. Xu et al. [Citation2015] compared women who had only G-CSF and G-CSF with endometrial scratching and concluded that endometrial scratch did not impair G-CSF treatment of thin endometrium and favored a higher clinical pregnancy rate.
We have concluded that the infusion of G-CSF leads to the improvement of endometrial thickness in comparison to the women who did not decide to have G-CSF. The secondary outcome measure of this study was the assessment of clinical pregnancy and live birth after frozen embryo transfer. The clinical pregnancy rate was 17.4%. and live birth rate only 6.89%. We did not observe significant differences between the two groups in baseline characteristics of the patients or the quality of transferred blastocyst thus leading us to the conclusion that even though the infusion of G-CSF improves the endometrial thickness it does not improve the clinical pregnancy rate. Further randomized studies with large numbers of patients are needed to confirm G-CSF usefulness in treatment of thin unresponsive endometrium.
Materials and methods
There were 134 patients with previously cancelled IVF cycles due to thin unresponsive endometrium (<7 mm) undergoing IVF in a frozen cycle at the INVICTA Fertility Clinic from October 6, 2011 until October 23, 2014. Patients fulfilling all the inclusion criteria were offered the G-CSF therapy. In the case of a positive decision, the patient was assigned to the treatment group, and in the case of a negative decision to the control group. Both groups of patients had two measurements of the endometrium thickness before FET.
The 62 treatment group patients, out of 134, fulfilled all the inclusion criteria: i) patients with previously cancelled IVF cycles due to thin unresponsive endometrium (<7 mm); ii) previous treatment of unresponsive endometrium with oral/vaginal estradiol, sildenafil citrate, or salicylic acid; iii) lack of contraindications for G-CSF treatment; and iv) undergoing IVF in a frozen cycle with own embryos. Twenty-nine of the women agreed to participate in the G-CSF therapy and received intrauterine G-CSF infusion. The control group consisted of the 33 women who declined the offer to participate in the study.
All the women underwent diagnostic hysteroscopy before entering the study. None of the patients had diagnostic curettage, endometrial ‘scratching’, or prolonged exposure to high doses of progesterone before the study. Additionally, none of the women had Asherman’s syndrome, fibroids, or polyps in diagnostic hysteroscopy performed prior to embryo transfer. We also excluded patients whose embryos were selected for transfer based on results of preimplantation genetic screening or diagnosis.
All procedures performed in this study were in accordance with 1964 Helsinki declaration and its later amendments and the study protocol was approved by the Institutional Review Board of Varmia and Masuria, Olsztyn, Poland, and written informed consent was given by each participating woman. Women from both the G-CSF and control groups had previously received a standard long protocol with the GnRH- agonist protocol- triptorelin acetate, at 0.1 mg/d s.c. (Gonapeptyl, Pharmacia Upjohn, Kalamazoo, MI, USA). Ovarian stimulation began 14 days thereafter with human menopausal gonadotropin hMG (Menopur, Ferring Pharmaceuticals, Copenhagen, Denmark). Ovulation was induced by administration of 5,000 IU hCG when the three leading follicles had reached a diameter of 17 mm. The oocyte retrieval procedure was performed 36 h after hCG administration (Pregnyl, Organon, Oss, The Netherlands) [Lukaszuk et al. Citation2005].
Embryos were cultured to the blastocyst stage and then vitrified using Cryotop (Kitazato Co. BioPharma, Shizuoka, Japan) according to the manufacturer’s specifications [Mukaida et al. Citation2001]. In preparation for transfer, frozen blastocysts were warmed according to the manufacturer’s specification with Cryotop Safety Kit Thawing (Kitazato Co. BioPharma) and cultured at 37oC for over 2 h in the G2 medium (Vitrolife, Sweden). Immediately after warming, blastocyst survival was evaluated according to morphologic appearance and the ability of the blastocele to re-expand before transfer [Gardner and Lane Citation1999]. Assisted hatching was performed in all cases [Gabrielsen et al. Citation2004]. For all patients, two blastocysts were transferred on day 5 of an estradiol with progesterone hormone replacement cycle. The preparation for FET began with a transvaginal ultrasound on the first or second d of menstrual cycle. Supplementation was started if estradiol was lower than 50 pg/ml and no ovarian cyst was found. Patients received daily 4 mg of vaginal estradiol (Estrofem Novo-Nordisk, Denmark). Additionally, each woman received low dose aspirin (100 mg/daily) and folic acid (0.4 mg/daily). After 7 d estradiol was increased to 6 mg/daily and after 2 more d, ultrasonography was performed. In all 62 cases the endometrium was below 7 mm (first measurement of endometrium). All women were offered the option to participate in the study and receive G-CSF infusion.
At this stage the 29 women who agreed to participate in the study received 30 mU (300 mcg/1ml) of G-CSF (Neupogen, Filgastrim; Amgev Inc., Thousand Oaks, CA, USA) under abdominal ultrasonography control. In all cases, Frydman embryo transfer catheters were used to perform the infusions. Endometrium was reassessed after 72 h (second measurement of endometrium). If it expanded >7 mm, luteal support was begun (a 3 × 200 mg/progesterone vaginally/daily). FET was performed on day 5 after the progesterone was first administered. In one case, at 72 h after G-CSF check, endometrium remained below 7 mm. The patient was presented with two options: continue with the transfer, despite inadequate endometrium, or cancel the cycle. She decided to continue with FET.
The control group consisted of 33 women. These patients also received 4 mg of vaginal estradiol which was increased to 6 mg after 7 d. The ultrasound after 2 additional d also showed endometrium below 7mm. As none of these women agreed to receive the G-CSF infusion they continued the 6 mg estradiol and their endometrium was also reassessed after 72 more h. At that point 17 women who still had endometrium below 7 mm decided to continue with the embryo transfer despite inadequate endometrium. All transfers were performed by two highly experienced doctors under ultrasound guidance.
The primary outcome was the endometrial thickness. The secondary outcome was clinical pregnancy and live birth. The endometrial thickness was measured in transvaginal sonography. The clinical pregnancy was defined as identification of an intrauterine gestational sac by transvaginal ultrasonography and live birth as the birth of a newborn, irrespective of the duration of gestation, that exhibits any sign of life, such as respiration, heartbeat, umbilical pulsation, or movement of voluntary muscles.
Statistics
Quantitative variables were evaluated for the normality of the data distribution using Shapiro-Wilk test and presented as medians (25th-75th percentiles) or means ± SD. Categorical variables were presented as ratios and analyzed using Chi square test. Differences between dependent and independent variables were compared by the Mann-Whitney-U nonparametric test or Wilcoxon test.
A value of p<0.05 was considered statistically significant. The statistical package STATISTICA (data analysis software system) version 10.0 (StatSoft Inc., Tulsa, OK, USA; www.statsoft.com) was used for data analysis. Due to the small number of patients included in the study we did not have any missing data.
Declaration of interest
The authors report no declarations of interest and no conflicts of interest.
Acknowledgments
The authors thank Justyna Stefaniak from Data Management and Statistical Analysis (DMSA) for data analyses. We thank Anna Knight for language support.
Additional information
Notes on contributors
Michał Kunicki
Conception and design of the idea, data interpretation, and preparation of manuscript: MK, KŁ, JL, PS, JSZ. All the authors conceived and planned the work that led to the manuscript or played an important role in the acquisition, analysis, and interpretation of the data. All the authors take responsibility for the work as a whole, from inception to the published manuscript.
Krzysztof Łukaszuk
Conception and design of the idea, data interpretation, and preparation of manuscript: MK, KŁ, JL, PS, JSZ. All the authors conceived and planned the work that led to the manuscript or played an important role in the acquisition, analysis, and interpretation of the data. All the authors take responsibility for the work as a whole, from inception to the published manuscript.
Joanna Liss
Conception and design of the idea, data interpretation, and preparation of manuscript: MK, KŁ, JL, PS, JSZ. All the authors conceived and planned the work that led to the manuscript or played an important role in the acquisition, analysis, and interpretation of the data. All the authors take responsibility for the work as a whole, from inception to the published manuscript.
Patrycja Skowrońska
Conception and design of the idea, data interpretation, and preparation of manuscript: MK, KŁ, JL, PS, JSZ. All the authors conceived and planned the work that led to the manuscript or played an important role in the acquisition, analysis, and interpretation of the data. All the authors take responsibility for the work as a whole, from inception to the published manuscript.
Joanna Szczyptańska
Conception and design of the idea, data interpretation, and preparation of manuscript: MK, KŁ, JL, PS, JSZ. All the authors conceived and planned the work that led to the manuscript or played an important role in the acquisition, analysis, and interpretation of the data. All the authors take responsibility for the work as a whole, from inception to the published manuscript.
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