Can Embryo Transfer With Self-spent Culture Medium Improve Clinical Pregnancy Outcomes in Vitro Fertilization - Embryo Transfer Process?
Phase: NA
Conditions: in Vitro Fertilization
Sponsor: Bin Wu

NCT03728140 
 
 
 
 
 
 
Study Protocol & 
Statistical Analysis 
 
 
 
 
 
 
 
 
 
 
12/01/2019 
 

 
Objective: 
A total of 335 couples participated in this study, which was performed between 
January 2018 and December 2019 at the Reproductive Medicine Department, Jinan 
Central Hospital Affiliated to Shandong University, and Reproductive Medicine Center, 
Gansu Provincial Maternity and Child-care Hospital. The couples underwent ICSI/IVF 
treatment and their high quality embryos were randomized to embryo transfer using 
either fresh or self-spent medium with informed consents and ethics approvals.  
Design: 
Patients met the following criteria were included: (i) ≤ 35 years old; (ii) regular 
menstrual cycles; (iii) baseline follicle stimulating hormone (FSH) <12 IU/L; 
(iv)infertility with simple tubal or/and male factors; (v) did not experience more than 
two prior failed ET cycles; (vi) at least two good-quality Day 3 embryo or one 
good-quality Day 5 blastocyst for ET. Exclusion criteria were as follows: (i) uterine 
abnormalities, such as adenomyosis, endometriosis and endometrial polyps; (ii) 
ovarian abnormalities, such as low ovarian response and polycystic ovary syndrome 
(PCOS); (iii) endometrial thickness <7 mm on transfer day. 
Methods: 
Ovarian stimulation and IVF/ICSI procedure 
Controlled ovarian hyperstimulation was carried out with uninterrupted 
injection of recombinant FSH (Gonal-F; Merck Serono; Switzerland) following 
pituitary down-regulation by GnRHa. When the dominant follicles measured ≥ 18 
mm in diameter, human chorionic gonadotrophin (hCG, Lizhu Inc, China) or 
recombinant hCG (Ovidrel, Merck Sereno, Switzerland) was administrated, and 
oocyte retrieval was performed approximately 36.5 h later. Sperms were prepared 
using density gradient centrifugation and swim-up techniques before fertilization, 
and IVF/ICSI procedures were applied 4–6 h after oocyte retrieval. Fertilization was 
assessed by checking the appearance of two pronuclei 16–18 h later. 

Embryo culture 
After fertilization, embryos were cultured one by one in G1TM-PLUS medium 
drops (Vitrolife, V.Frölunda, Sweden) containing human serum albumin and 
gentamicin covered with OVOILTM (Vitrolife, V.Frölunda, Sweden) in the culture dish 
until Day 3, G2TM-PLUS medium was sequential used from Day 3 to Day 6 if the 
embryos need to blastocyst culture. All mediums for embryo culture were 
pre-equilibrated at 37°C and 6% CO2 before use.  
Cleavage stage embryos were morphologically classified according to the 
number and shape of blastomeres, the percentage of fragmentation as well as 
multinucleation on Day 3, as following: Grade 1, no or <5% fragmentation with at 
least six equal‑sized blastomeres and no multinucleation; Grade 2, <20% 
fragmentation with slightly unequal‑sized blastomeres and no evidence of 
multinucleation; Grade 3, 20%–50% fragmentation with obviously unequal‑sized cells; 
and Grade 4, >50% damage or severely unequal‑sized cells or visible multinucleation. 
Blastocysts were classified according to development stage (staging 1-5) and 
morphology of inner cell mass (Grading A-C) and trophectoderm (Grading A-C).  
Embryo transfer 
Two Day 3 fresh cleavage stage embryos scored better than grade 2 or single 
Day 5 fresh blastocyst stage embryo scored better than 4BB was selected for 
intrauterine transfer. The selected embryos were pre-transferred into a 500 μl 
pre-equilibrated fresh culture medium (G1TM-PLUS or G2TM-PLUS) awaiting ET. In the 
test group, the stretched thin Pasteur pipettes were used to absorb the self-spent 
droplets under the oil, and the droplets were transferred at least three times in a 
new dish to remove the oil floating on the surface. Then, the selected embryos were 
transferred to the combined droplet for catheter loading. Correspondingly, the 
embryos of the control group were loaded directly in the pre-equilibrated fresh 
culture medium. Luteal support by progesterone administration was provided to the 
patients after transfer. 
Outcome Measures 

Chemical pregnancy was confirmed by detecting serum β-hCG level 14 days 
after embryo transfer, and clinical pregnancy was defined as presence of at least one 
gestational sac identified by ultrasound 5 weeks after transfer. Biochemical 
pregnancy rate was defined as the number of early pregnancy losses divided by the 
total number of positive serum β-hCG. Clinical pregnancy rate was defined as the 
number of clinical pregnancies divided by the number of transfer cycles. 
Implantation rate was calculated as the number of geastational sacs divided by the 
number of embryos transferred. Clinical abortion rate was defined as the number of 
clinical pregnancy losses before 28 weeks of gestation divided by the total number of 
clinical pregnancies. Live birth rate was defined as a ratio between healthy living 
newborns and embryo transfer cycles.  
Statistical Analysis: 
Data were analyzed using student’s t-test and chi-square test. P<.05 was 
considered statistically significant.