Study design and patients
This retrospective study was conducted at the Center for Reproductive Medicine of Yantai Yuhuangding Hospital and included women admitted from January 2017 to September 2018. The inclusion criteria were: 1) infertility; 2) underwent IVF treatment; and 3) hysteroscopy was performed within 3 months prior to IVF in order to diagnose CE according to Delphi’s diagnostic criteria 28. The exclusion criteria were: 1) dilated endometrial vessels; 2) refused hysteroscopy; or 3) not suitable for hysteroscopy (suspected acute reproductive infection, menstruation at the time of examination, unexplained uterine bleeding, or a positive result on a pregnancy test).
In order to exclude possible confounding factors that might influence the pregnancy outcomes, such as age, cause of infertility, combined disease, follicular stimulation protocol, and quality of the transplanted embryos, a group of standardized patients was selected from the participants enrolled in this study according to the following standards: 1) <35 years of age; 2) normal ovarian reserve function (antral follicle count >7, anti-Müllerian hormone level of 1.0–4.0 ng/mL, and baseline follicle stimulating hormone [FSH] level <10 IU/L) 36; 3) standard long-term protocol for follicle stimulation; 4) fallopian tube obstruction as the single infertility factor for IVF treatment; and 5) underwent fresh embryo transfer. The exclusion criteria were: 1) endometrial carcinoma; 2) comorbidities including thyroid disease, hypertension, diabetes, and hematologic diseases; 3) hyperprolactinemia; 4) cervical lesions or conization of the cervix; 5) intrauterine devices; 6) uterine fibroids; 7) autoimmune diseases; 8) chromosomal abnormalities in either male or female family members; 9) recurrent abortions; 10) congenital uterine malformations; 11) pelvic or genital tuberculosis; and 12) uterine scarring.
This study was approved by the ethics committee of Yantai Yuhuangding Hospital. All data were extracted from the database of the Center for Reproductive Medicine of Yantai Yuhuangding Hospital. All methods were performed in accordance with the relevant guidelines. The patient’s informed consent was obtained for inclusion in the database.
The included patients were divided into the CE group (diagnosed with CE) and non-CE group (not diagnosed with CE) based on the hysteroscopy findings. Then, the patients with CE were divided into five subgroups according to the hysteroscopic characteristics: hemorrhagic spots, hyperemia (including diffuse hyperemia and focal hyperemia), micropolyps, hyperemia combined with micropolyps, and other.
The diagnostic criteria for CE at hysteroscopy were based on Delphi′s diagnostic criteria 28: 1) diffuse hyperemia: large areas of hyperemia with white points (Figure 1A); 2) focal hyperemia: small areas of hyperemia (Figure 1B); 3) hemorrhagic spots: focal red areas with sharp and irregular borders possibly in continuity with a capillary (Figure 1C); 4) micropolyps: endometrial polyps <1 mm in diameter with prominent vascular pedicles, distributed focally (Figure 1D) or diffusely (Figure 1E); and 5) stromal edema: thick and pale appearance of the follicular endometrium (a normal finding during the secretory phase, Figure 1F). The diagnosis and classification of CE were performed by two physicians who had received professional training.
The histopathologic diagnostic criterion for CE 6 was the detection of plasma cell-specific surface antigen CD138 by immunohistochemistry. CE was diagnosed if at least five plasma cells were counted in the endometrial stroma in each randomly chosen high-magnification field (×400) using an Olympus (Tokyo, Japan) microscope.
Management of CE
The patients with CE scheduled for frozen embryo transfer (FET) received treatment for CE: oral doxycycline 100 mg bid plus metronidazole tablets 0.4 g tid for 2 weeks, or cefdinir dispersible tablets 100 mg tid orally plus oral metronidazole tablets 0.4 g tid for 2 weeks. At the same time, some patients used traditional Chinese medicine enema for 10 days after the end of menstruation and for two consecutive menstrual cycles. The main treatment to improve the endometrial receptivity of patients with CE who were scheduled for FET was down-regulation and induction of an artificial cycle. The patients with CE who were scheduled for fresh embryo transfer were advised to undergo whole embryo freezing and then to undergo frozen embryo transplantation after anti-inflammatory therapy for CE mentioned above. For the patients with CE who were scheduled for fresh embryo transfer but refused anti-inflammatory therapy, or the patients with CE who had mild inflammation that did not qualify for anti-inflammatory treatment mentioned above, prophylactic antibiotic treatment (second-generation oral cephalosporin tid for 3 days) after hysteroscopy was used.
Before IVF treatment, all patients with CE were fully informed regarding the inflammatory status of their uterine cavity. With the informed consent of the patient, whole embryo freezing was performed after oocyte retrieval to allow for anti-inflammatory treatment for CE to be administered. If anti-inflammatory treatment was refused or not recommended due to mild inflammation, embryo transfer in the cleavage stage was performed on the third day after oocyte retrieval. The gonadotropin-releasing hormone (GnRH) agonist regimen was used as the standard long-term protocol and involved the daily injection of 0.05 mg triptorelin acetate (Ipsen; Boulogne-Billancourt) in the mid-luteal period of the preceding menstrual cycle. Pituitary suppression (luteinizing hormone level <5 IU/L, estradiol level <50 ng/L, endometrial thickness <5 mm, and no functional ovarian cyst) was achieved after 14 days. The dose of recombinant FSH (Gonal F; Serono, Rockland, MA) or purified urinary human menopausal gonadotropin (Repronex; Ferring Pharmaceuticals, Suffern, NY) was adjusted (75–300 U/d) to achieve ovarian stimulation. When at least one follicle was >17 mm in diameter, 4000–10000 IU of human chorionic gonadotropin (hCG) was administered subcutaneously, and ultrasound-guided transvaginal oocyte retrieval was performed 35 hours later. Embryo transfer in the cleavage stage was performed on the third day after oocyte retrieval. All patients undergoing fresh embryo transfer received luteal support until pregnancy. The dosing was stopped after 10 weeks. Serum hCG level was measured 14 days after embryo transfer, and ultrasound was performed 28 days after embryo transfer.
Hysteroscopy and endometrial biopsy
Since there is some evidence that hysteroscopy can improve outcomes after ART (Di Spiezio Sardo et al., 2016), our center recommends that all patients undergo hysteroscopy before IVF. Hysteroscopy was scheduled for day 6–12 of the menstrual cycle. The procedure was performed using a rigid hysteroscope with a 3.5-mm-diameter outer sheath and a 30° viewing angle (Karl Storz, Germany). Saline (0.9%) was used as the medium at 100 mmHg pressure. All hysteroscopies were performed by two physicians who had received professional training. The video results were recorded in the MEDCON medical information technology network system. For patients who consented, the endometrium was sampled blindly at the end of hysteroscopy using a metal curette for endometrial biopsy. All patients received prophylactic oral antibiotic therapy ( Cefuroxime ester tablets 250mg bid) for 2 days after hysteroscopy.
The clinical data of all patients were retrieved from the Wuhan Mutual Creation Assisted Reproductive Information Management System, including age, infertility duration, type of infertility, cause of infertility, initial diagnosis, body mass index (BMI), ovarian reserve function, mode of ART, indications, medication protocol, oocyte retrieval, embryo transfer, frozen embryo condition, FET, clinical pregnancy rate (CPR), live birth rate (LBR), premature birth rate, and miscarriage rate. Previous medical records, including past medical history, obstetric history, fallopian tube examination findings, and endometrial histology results, were also retrieved from Wuhan Mutual Creation Assisted Reproductive Information Management System. The primary outcome of the study was CPR, and secondary outcomes were LBR, premature birth rate, and miscarriage rate.
A high-quality embryo was defined as a grade 1–2 embryo comprising 7–9 cells 37. Blastocyst quality was evaluated according to the Gardner scoring system 7, and high-quality blastocysts were defined as having embryo scores greater than 3BB, which excluded the inner cell mass and trophoblastic layer C. Clinical pregnancy was defined as one or more pregnancy sacs identified during ultrasonography. Biochemical pregnancies were not included 36. The CPR was defined as the number of clinical pregnancy cycles/number of embryo transfer cycles × 100% 36. The LBR was defined as the number of live births/number of embryo transfer cycles × 100% 36. The miscarriage rate was defined as the number of cycles with spontaneous abortion within 28 weeks/number of clinical pregnancy cycles × 100% 36. The premature birth rate was defined as the number of birth cycles within 28–37 weeks/number of clinical pregnancy cycles × 100% 36.
All patients were followed-up by nursing staff. Blood hCG level was measured 14 days after transplantation. A vaginal ultrasound examination was performed 28 days after transplantation. Ultrasound was repeated at 10 weeks of pregnancy. Follow-ups were conducted during mid-pregnancy, late pregnancy, and after childbirth by telephone to record any comorbidities.
All data were analyzed using SPSS 21.0 for Windows (IBM, Armonk, NY, USA). Continuous data were tested for a normal distribution using the Kolmogorov-Smirnov test. Normally-distributed continuous data are presented as means ± standard deviations and were analyzed using the t-test for independent samples or single-sample ANOVA. Non-normally-distributed continuous data are presented as medians (ranges) and were analyzed using the Wilcoxon rank-sum test. Categorical data were analyzed using the chi-squared test, Fisher’s exact test, or the corrected chi-squared test, as appropriate. Factors associated with pregnancy outcomes were identified using univariable and multivariable logistic regression analysis with stepwise selection, and odds ratios (ORs) and 95% confidence intervals (95%CIs) were calculated. P<0.05 was taken to indicate statistical significance.