All through the world, about 15% of couples struggled with infertility (1). Assistance Reproductive Technology (ART) can be so helpful to solve this problem but Several factors can affect in vitro fertilization (IVF) success rates. The most important independent variables are woman’s age, anti-Müllerian hormone (AMH) level, and number of transferred embryos and their quality (2-4). Studies have also shown that an endometrial thickness (ET) of less than 7 mm negatively influences pregnancy outcomes (5, 6).Further, some evidence suggests that immunological mechanisms in the endometrium are critical to the implantation process (7). Some researchers have noted that growth factors, hormones, and cytokines synthesized by decidual cells are involved in the implantation process (8).
Previous studies have demonstrated that granulocyte colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of neutrophilic granulocytes, affects macrophages in decidual cells, and ultimately influences the implantation (9).
Recent studies have indicated that intrauterine injection of G-CSF is effective in treatment-resistant infertile women (10, 11). G-CSF is a cytokine that stimulates the proliferation and differentiation of neutrophilic granulocytes (12).
This drug improves fetal implantation, affects decidual macrophages, ovulation, ovarian function, and granulosa cell function, boosts the ovarian response to gonadotropin in patients with a poor response, and reduces recurrent miscarriages. It is also involved in the recovery of primary endometrial lesions and suppresses autoimmune reactions (13-21).
In addition to what was noted, G-CSF as a biomarker for the implantation potential of an egg or embryo can predict IVF outcomes (22, 23).
Given the above-mentioned description and considering the limited studies evaluated the effects of intrauterine injection of G-CSF, in this study, we attempt to evaluate and compare the effects of intrauterine and subcutaneous injections of G-CSF on increasing ET and pregnancy outcomes in infertile women who were candidates for IVF.

 

This double-blind trial study was conducted on 34 women who were candidates for frozen IVF cycles in Yas Hospital. This study was approved by the Ethical Committee of Tehran University of Medical Sciences (ethical code: IR.TUMS.MEDICINE.REC.1396.28 11), and all patients signed informed consent.
Patients with a history of failed cycles and ET of less than 7 mm (measured by ultrasound) were included based on the inclusion and exclusion criteria. In the current study, women aged 18 to 40 years, who had at least two failed IVF cycles with a minimum of three suitable embryos for transfer, were enrolled.
Patients were excluded from the study in the case of having contraindications for G-CSF injection, suffering from any systemic diseases, dealing with Asherman’s syndrome, fibroids, and polyps detected by hysteroscopy, and being unwilling to participate in this study. After entering the study, the patients were divided into two groups using a random number table method.
At the beginning of the study, all the patients underwent laboratory tests, including fasting glucose sugar, lipid profile, and liver and thyroid function tests, to rule out systemic diseases.
Estradiol valerate tablets (2 mg; Aburaihan ‌Pharmaceutical Company, Tehran, Iran) were administered daily for all the patients from the second day of the menstruation cycle, and then the estradiol dose was increased to 10 mg per day. On the 12th day of the menstruation cycle, transvaginal ultrasound was performed, and ET was measured in the thickest part and the longitudinal view. In the case of thickness less than 7 mm, ultrasound was again conducted on the same day by another radiologist, and the mean values ​​measured as ET were recorded in the patient’s medical file, and the patient was treated with G-CSF.
In the first group, 300 μg of G-CSF (Neupogen, F.Hoffmann-LA Roche, Swiss) with a volume of 1 mLcc was transvaginally injected into the uterus by intrauterine insemination (IUI) catheters. For making it a double-blind study, a placebo (normal saline with a similar volume) was administered simultaneously and subcutaneously. In the second group, the same dose of G-CSF (300 μg per 1 mLcc) was injected subcutaneously, and the intrauterine placebo was injected. All the drugs were prepared and coded for injection blindly.
ET was assessed by ultrasound 72 hours after G-CSF injection. All the patients’ sonographies were performed by a radiologist between 9 am AM and noon, who did not know the grouping and method of drug injection. If ET was more than 7 mm, after receiving progesterone for three days, a three-day embryo was transferred. In the case of no increase in ET (above 7 mm), the cycle was canceled.
The rate of chemical pregnancy in the two groups was evaluated two weeks after the embryo transfer by measuring the serum β‑subunit human chorionic gonadotropin BHCG levels (using Roche kit, electrochemiluminescence immunoassay )ECLIA( method, and Cobas  411 device, considered positive when the value was more than 25). The rate of successful embryo implantation was tested four weeks after the embryo transfer by transvaginal sonography, and the clinical pregnancy evaluations were performed six weeks after the embryo transfer by abdominal sonography.