Patients.
This study was a retrospective research of patients who came to our center for IVF treatment. The present study was approved by the institutional ethics committee review board of the Affiliated Yuhuangding Hospital of Qingdao University, Shandong, China and all methods were carried out in accordance with relevant guidelines and regulations. From January 2012 to December 2019, a total of 18 patients with conjoined oocytes were included in this research. All patients gave written informed consent prior to the start of the study. The patient's age ranges from 24 to 43.
Ovarian stimulation and oocyte retrieval.
Most patients used the long protocol were down regulated with GnRH agonist triptorelin(Decapeptyl; lpsen-biotech, Paris, France), then administered with follicle-stimulating hormone(Lizhu Group Co., Ltd, Zhuhai, China; Merck Serono, Geneva, Switzerland) daily on the basis of patient’s age and ovarian reserve. Other protocols were also adopted in ovarian stimulation, such as antagonist protocol or mini-stimulation protocol according to the patients’ response to stimulation. To induce the oocyte maturation, recombinant human chorionic gonadotrophin (Merck Serono, Geneva, Switzerland) or HCG (Lizhu Group Co., Ltd, Zhuhai, China) was injected when ≥ 3 follicles reached the diameter of 17–18 mm. After HCG injection, oocytes were collected at 36-38h and cultured at incubators until insemination.
Sperm manipulation and insemination.
The semen was collected by masturbation after 2–7 days of abstinence and liquefied at room temperature for 30min. After liquefaction, the sperm suspension was mixed and a 5µL aliquot was loaded into a Makler chamber. Semen parameters (concentration and motility) were assessed under a Makler-chamber. Sperm was collected by conventional discontinuous density gradient centrifugation and swim-up method according to the World Health Organization (WHO) guidelines and then stored in an incubator for subsequent insemination. Oocytes were incubated in culture medium for 2-3h after being picked up and then incubated with motile sperm. The oocytes were incubated at a concentration ratio of 50 000 sperm/ml.
Fertilization assessment and embryo culture.
Fertilization was evaluated by the formation of pronucleus approximately 17-18h after insemination. Oocytes with two pronuclei were considered normal fertilization, and oocytes with one or more than two pronuclei were considered abnormal fertilization. Embryo culture was carried out according to the operating instructions of Vitrolife(Vitrolife, Gothenburg, Sweden). Briefly, the activated oocytes would be cultured in GI medium for subsequent development. On day 3, the embryo quality score was carried out, and the embryos with 7–9 symmetrical blastomeres and the fragmentation rate of less than 10% were rated as high-quality embryos. Embryos were transferred into GII medium on the afternoon of day 3 for further development. Blastocyst formation score was performed on days 5 and 6, only blastocysts with dilated blastocyst cavity, clearly visible inner cell masses and compact trophoblast cells were considered as available blastocysts.
Clinical pregnancy.
On the third day after oocyte retrieval, 1–2 good quality embryos were transplanted under ultrasound guidance. The embryo transfer tube (Cook, Ireland Ltd, Ireland) was used to embryo transfer (ET). After ET, the tube was examined to make sure that there were no retained embryos. All ET patients were administered duphaston or injected progesterone for luteal support. About seven weeks after embryo transfer, patients were checked for clinical pregnancy by the presence of a gestational sac. The clinical pregnancy rate was defined as the number of clinical pregnancy divided by the number of patients.
Measurement of area and zona pellucida thickness of conjoined oocytes.
The area and ZP thicknesses of conjoined oocytes were measured on Day 0. All area and ZP thickness assessments were performed directly under an inverted microscope equipped with Hoffman modulation contrast optics. Oocytes images were recorded using a colour digital camera mounted on an inverted microscope by Hoffman modulation contrast. The setting for microscopic observations(200× magnification) and bright fields were kept constant throughout the study. Clear images of oocytes stored on the computer were displayed. The area and ZP thickness were measured using a public domain Image J program and ZP thickness was measured by selecting 4 sites on zona pellucida as 0:00, 3:00, 9:00 and 12:00. The calculated average ZP thickness is ZP average = (ZP1 + ZP2 + ZP3 + ZP4)/4. To minimize experimental distortion, the same technician performed all image recording and measurements.
Statistical analysis.
The software used was Statistics Package for Social Science (SPSS22; SPSS Inc., Chicago, IL, USA). Percentage data were arc sine transformed and analyzed with ANOVA when each measure contained more than two groups or with an independent t-test when each measure had only two groups; a Duncan multiple comparison test was used to locate differences. Categorical variables were expressed as the percentage. P < 0.05 was considered significant.