The retrospective analysis was performed on patients who underwent IVF treatment/intracytoplasmic sperm injection (ICSI) and fresh embryo transfer at Chenggong Affiliated Hospital of Xiamen University between January 2013 and December 2015. Institutional review board approval for this retrospective study was obtained from the Ethical Committee of the Medical College of Xiamen University (2018-023). Informed consent was overridden by ethics comment because the research was based on unidentifiable records. All research was conducted in accordance with relevant guidelines/regulations.
Only patients undergoing conventional ovarian stimulation (agonist or antagonist) were reviewed. Patients on light stimulation cycles, natural cycles and luteal phase stimulation cycles were excluded from the study (n=177). Forty-eight transfer cases lacked embryo transfer depth recording and were therefore excluded from the study. We also excluded patients identified as difficult to transfer (n = 100) and patients who had bacterial infections after transfer (n = 3). In none of the cases examined was there ever a case of blood in the catheter. Details of patient inclusion are shown in Fig. 1 additional.
Stimulation protocols and laboratory procedures
In all stimulation cycles, patients received 2-3 vials (75-225 IU) of gonadotropin daily during gonadotropin stimulation. Initial and continuing dosage was adjusted based on patient age, antral follicle count (AFC), body mass index (BMI), and follicle growth response. Recombinant follicle-stimulating hormone (FSH) (Gonal-F; Merck-Serono, Switzerland) or domestic urinary HMG (HMG; Lizhu, China) has been used for gonadotropin stimulation. During treatment, ovarian response was monitored by transvaginal ultrasound measurements of follicle growth and serum E2 level every 1 to 3 days. Gonadotropin stimulation continued until ultrasound revealed at least one follicle measuring ≥ 18 mm in mean diameter. 5,000 to 10,000 IU of human chorionic gonadotropin (hCG; Lizhu, China) was injected intramuscularly. Endometrial thickness and endometrial ultrasound pattern (Pattern A: a three-line pattern consisting of a central hyperechoic line surrounded by two hypoechoic layers, Pattern B: an intermediate isoechoic pattern with the same reflectivity as the surrounding myometrium and an ill-defined central echogenic pattern and pattern C: homogeneous and hyperechoic endometrium) were also assessed on the day25. Oocyte retrieval was scheduled 34 to 36 h after hCG administration and performed under transvaginal ultrasound guidance.
The oocytes were inseminated by conventional IVF or ICSI. Pronuclei were identified 17–18 h later. On day 3, embryos were given quality scores and embryos were assessed for cell number and size and degree of fragmentation. For patients receiving blastocyst transfer, the Gardner Scale26 was used to assess embryo quality. High quality embryos for transfer were defined as: embryos with less than 10% fragments and in-time cell size on day 3 and good internal cell mass and trophectoderm on day 5.
Fresh embryo transfers were performed on day 3 or day 5. Patients decided on the day of embryo transfer after clinical consultation. The number of embryos transferred varied from 1 to 3 depending on national regulations27. Transfer of three embryos was considered only in women with advanced age or repeated failures, and no patient had more than two blastocysts transferred.
All transfers were performed in the same room by seven experienced clinicians. Patients undergoing transfer received a transfer simulation the day before the embryo transfer. All patients were placed in the lithotomy position during the transfer procedure and the cervix was exposed using a bivalve speculum. The external os was cleaned with saline solution and the cervical mucus was removed with a cotton swab.
The Cook catheter outer catheter (K-JETS-7019-SIVF, Cook, IN, USA) was inserted under the guidance of abdominal ultrasound. The embryos were loaded into the internal catheter by the “three-drop technique”28. The drop of medium containing the embryos was separated from a previous drop and a following drop of medium by an air bubble, and the volume of the air bubble and the drop did not exceed 10 µL.
Embryos were injected with medium and air bubbles into the uterine cavity at low speed under ultrasound guidance. The injection position was addressed to the thickest part of the endometrium possible29. The bubble generated after the transfer was visualized under ultrasound and the distance between the position of the bubble and the fundal myometrium-endometrium interface was used as a marker of the position of the embryo (depth of embryo transfer). The catheter was then gently removed and examined under a stereomicroscope to ensure that all embryos had been transferred. After the transfer, the patients remained in bed for 30 minutes.
Luteal phase support was maintained with natural progesterone in oil (progesterone; XianJu, China), 60 mg im daily from the day of oocyte retrieval. A pregnancy test (determination of serum β-hCG) was performed 14 days after embryo transfer. Clinical pregnancy was defined as the presence of one or more gestational sacs detected on ultrasound performed 4 weeks after embryo transfer. If no signs of an intrauterine gestational sac were detected after an elevation of β-hCG, the ectopic pregnancy was confirmed by surgical treatment.
For data analyses, transfer depth was aggregated across transfer cycles into quartiles. In order to test the effect of extreme values, 10% of the percentile and 90% of the distance were also used as categorization criteria in the multivariate analyses.
For continuous variables, QQ plots were used to graphically assess the normality of the distribution. The distribution was considered normal when the plot was close to a straight diagonal line. One-way analysis of variance (ANOVA) for normally distributed data and Kruskal Wallis test for non-normally distributed data were used for the analyses, respectively. Categorical variables were presented as proportions and percentages of the total. Dichotomous variables were analyzed by chi-square test or Fisher’s exact test, as appropriate. When the test was significant (P
To perform multivariate analyses, the Generalized Estimating Equations (GEE) model was used because a patient may receive multiple transfers in the study. Multivariate analyzes were performed to assess the association between the depth of embryo transfer and the likelihood of clinical pregnancy, with adjustment for significant confounding factors. Transfer depth was assessed either as a categorized value mentioned above or as a continuous value (per millimeter increased) in the multivariate analyses. Covariates were selected based on their clinical importance. The model included patient characteristics known to be important for IVF outcome counseling, such as age, BMI, AFC, live births or previous pregnancies, duration of infertility, and the etiologies of infertility.30. Stimulation characteristics, including stimulation dose, gonadotropin-releasing hormone (GnRH) analogs used31the number of oocytes11endometrial thickness and shape25and elevation of progesterone on the day of induction21 were also selected because they are known to influence results. Finally, the model was also checked for other factors that may affect the outcome of the embryo transfer, including the stage of development of the transferred embryos, the presence of at least one good quality embryo transferred, and the different clinicians who performed the embryo transfer.
To explore whether covariates correlated with uterine contraction modified the effect of embryo transfer depth, interaction terms were introduced into the model. Interactions between embryo transfer depth and blastocyst transfer24elevated progesterone22and endometrial thickness23 were studied on the basis of previous knowledge. For ease of analysis, endometrial thickness on the day of hCG was categorized into thin categories ( 11mm). Median values in each transfer depth category were included as a continuous variable to test the overall linear trend across quartiles (p for trend).
All calculations were performed with SPSS (version 19; IBM). In all analyses, P