Hospital settings and patients inclusion
This is a retrospective cohort study conducted at the locally largest Reproductive Center. Consecutive patients requiring transvaginal ultrasound guided oocytes retrieval by a single operator were recruited from October to December 2019. The operator had 3-year experience in sonography and reproductive medicine, but no previous experience in oocytes retrieval procedure. Every patient signed informed consent to the collection of their clinical data for research use. The study protocol was approved by the hospital ethics committee (No. 2020-037). All procedures performed in the study were in accordance with institutional standard operation procedure (SOP) and ART regulation.
Data from patients met the following criteria were excluded. 1) Poor responders were excluded based on Bologna criteria (Women who followed at least 2 of the following 3 criteria. Women aged ≥ 40 years old, women with previous poor response with no more than 3 oocytes retrieved using a conventional stimulation protocol, and women with abnormal ovarian reserve test with antral follicle counting number (AFC) < 7 follicles and anti-Mullerian hormone (AMH) < 1.1 ng/ml . 2) Hyper-responders, determined as women with ≥ 15 oocytes to be retrieved .
The demographic data of all patients including female age, male age, infertile duration, infertile type and factors, body mass index (BMI), AFC, and AMH were collected.
Standard ovarian stimulation protocols were provided based on women’s age, ovarian reservation function, ovarian response in the previous cycles, patients’ and physicians’ preferences. Majority of women received ovarian stimulation using gonadotropin releasing hormone (GnRH) agonist and antagonist protocols. Otherwise, microflare or luteal phase stimulation were provided. Gonadotropin (Gn) were administrated using follicle-stimulating hormone (FSH, either Gonal-F, Merck Serono, Modugno, Italy, or Puregon, MSD Organon, Oss, Netherlands), human menopausal gonadotropin (HMG, Livzon, Zhuhai, China), or luteinizing hormone (LH, Luveris, Merck Serono, Modugno, Italy). Follicle growth was monitored regularly using transvaginal ultrasound. When at least two follicles reached an average diameter at 18 mm, or at least three follicles reached an average diameter at 17 mm, human chorionic gonadotropin (HCG, Merck Serono, Geneva, Switzerland, or Livzon, Zhuhai, China) were used for triggering of ovulation.
The single trainee performed oocyte retrieval under the monitoring of a 7-year experienced tutor. The tutor was responsible for the supervision of the trainee during the entire learning period, until the trainee had the final assessment of technique proficiency examination after performing about 300 cases of oocyte retrieval.
Oocytes were aspirated about 36- to 37-hour following HCG triggering. The whole procedures were conducted under general anesthesia (Propofol, Libang Pharmaceutical, Xi’an, China) or with the help of pain relief drug (Ibuprofen, SmithKline & French Laboratories Ltd., Tianjin, China). The aspirations were conducted using an 18-gauge needle under vacuum and guided by transvaginal ultrasound monitoring. The vacuum suction pressure was set to about 150–160 mmHg, and kept constant pressure during the procedures. Follicles with estimated size ≥ 10 mm were aspirated. The aspirants were transferred to the embryologist immediately, and the recovered oocytes were counted and further inseminated with sperms through either in vitro fertilization (IVF) or intra-cytoplasmic sperm injection (ICSI) based on the partners’ sperm quality.
At the end of oocytes retrieval, a vaginal checking with gauze was performed to prevent or stop the oozing at puncture sites. If the vaginal bleeding cannot be stop within around one minute. One or two gauze were packed inside vaginal for vaginal compression to stop the bleeding. Patients with vaginal compression to stop prolonged vaginal bleeding at puncture sites were also recorded and marked as “vaginal bleeding” in this study.
Up to 2 embryos were transferred at either cleavage stage or blastocyst stage based on the number and quality of embryos and the age of female patients. The remaining usable embryos were cryopreserved by vitrification.
The primary outcome measured of the study was operative time and oocytes retrieval efficiency. The operative time was used for determining the learning curve, and defined as the time duration between the operator started the first puncture and operator finish the vaginal checking. The oocytes retrived efficiencies were measured by dividing the number of oocytes retrived by the number of oocytes expected as determined by transvaginal ultrasound on trigger day, and were calculated based on the following methods. Method 1: Oocytes retrived efficiency (%, ≥ 10 mm) = number of oocytes retrived/number of follicles sized ≥ 10 mm on trigger day * 100%. Method 2: Oocytes retrived efficiency (%, ≥ 14 mm) = number of oocytes retrived/number of follicles sized ≥ 14 mm on trigger day * 100%.
The secondary outcomes assessed here including number of oocytes retrieved, fertilization rate, number of usable embryos on day 3, and number of embryos with top quality. The fertilization rate was evaluated as the proportion of 2 pronucleus oocytes out of number of oocytes inseminated or MII injected based on either IVF or ICSI performed. Embryo quality was evaluated on day 3 based on the assessment of blastomere number, extent of fragmentation and the symmetry of blastomere. The total score of each embryo were calculated by the sum of above parameters’ evaluation as reported . Usable embryos on day 3 were those with > 5 blastomere, < 50% fragmentation and an overall score > 5 . Quality of blastocysts was evaluated based on the Gardner grade . Embryos with top quality were evaluated on day 3, and those with 8 to 10 regularly formed blastomere, no more than 20% fragmentation were marked as top quality.
Reproductive outcomes including number and reasons of cancelled embryo transfer cycles, stage and number of embryos transferred, clinical pregnancy rate, biochemical pregnancy rate, miscarriage rate, ectopic pregnancy rate, ongoing pregnancy rate were also measured. Clinical pregnancy rate was calculated as the proportion of patients with clinical pregnancy, who showed gestational sac on gestation 5 to 6 weeks, out of patients with embryos transferred. Miscarriage rate was calculated as the proportion of patients with spontaneous miscarriage out of patients with clinical pregnancy. Biochemical pregnancy rate was defined as the proportion of patients with biochemical pregnancy, who showed positive serum HCG levels but no gestational sac under sonography examination, out of the patients with embryo transfer. Ectopic pregnancy rate was defined as the proportion of patients with ectopic pregnancy whose gestational sac reside out of intrauterine cavity, out of those with embryo transfer. Ongoing pregnancy rate was defined as the proportion of patients with the presence of viable intrauterine fetus at gestational 14 weeks as confirmed by ultrasound out of those with embryo transfer.
The safety outcome was the number of vaginal bleeding at puncture site. No other complications, for instance, intraperitoneal bleeding, infection, organ injuries, occurred during the study period.
All statistical analyses were processed using SPSS 19.0 (Chicago, Illinois, US). Continuous data are demonstrated as mean ± standard deviation (SD) if they followed a normal distribution, and median and interquartile range (IQR) if not. Categorical data are demonstrated as numbers and proportions. Statistical analyses were performed using one way ANOVA for normally distributed continuous data. Nonparametric Kruskal-Wallis tests were used for data not following normal distribution. Chi-square tests and Fisher’s exact test were used to compare the categorical data. A P value < 0.05 was considered as statistically significant.
A CUSUM method was performed to determine the changing trend of operative time across the case series. The mean operative time was selected as a reference value. The sequential differences between the mean operative time and each individual operative time (DOT) were calculated. Then the CUSUM for each case was calculated as summation of DOT of this case and the previous sum of operative time differences. These summations of case series yield a curve showing the trend of change in operative time, and help to determine the critical points when a change of learning phase occurs. Therefore, the learning curve based on the operative time was demonstrated by plotting the CUSUM outcomes. Multiple linear regression analyses were performed to determine the predictor of oocytes retrieval efficiencies. A stepwise regression approach was used to eliminate the inter-relationship among the independent variables. Possible confounders (phases of learning curve, female age, AFC, AMH, total Gn days for ovarian stimulation, total Gn doses for ovarian stimulation, endometrial thickness on trigger day, operative time, etc) which might affect the oocytes retrieval efficiencies were introduced into the regression analyses.