Injection of Day 2 Embryo Culture Supernatant Into the Uterine Cavity Didn't Improve Implantation and Pregnancy Rates
Condition: Infertility; In Vitro Fertilization
MeSH terms: Infertility
Study type: Interventional
Phase: Phase 1

Summary: 
      Forty-five infertile women undergoing in vitro fertilization treatment were managed by
      uterine cavity injection of day 2 embryo culture supernatant before day 3 embryo transfer. No
      statistically significant differences were found in implantation and pregnancy rates when
      compared with control group.
    

Detailed description: 
      Study design and sample size This was a randomized clinical trial involving ninety patients
      who were undergoing conventional IVF or intracytoplasmic sperm injection (ICSI) treatment at
      our Reproductive Medical Center. The subjects were recruited and studied for a period of 6
      months between January and June 2008. The study was approved by the Ethics Committee of
      Shen-Zhen Maternity and Child Healthcare Hospital. Subjects all signed an informed consent
      form.

      According to the guideline made by the Ethics Committee of Shen-Zhen Maternity and Child
      Healthcare Hospital, we had better accomplish the study within six months. We calculated
      that, in our center, a sample size of 100 or more subjects would need to be recruited over
      about a 6 months period. We ended up with 90 patients in this study.

      Subject Ninety infertile women, aged 23-39 years, with an infertile duration of 3-8 years,
      were enrolled from our infertility treatment clinic between January and June 2008. The
      selection criteria for participants were only those women who were first going to undergo IVF
      or ICSI treatment. Women who were treated with natural cycle IVF or had no appropriate embryo
      for ET were excluded. The infertility factors were identified as the following: tubal (n=54),
      unexplained (n=3), male n=22), endometriosis (n=9), others (n=2). The selection and
      enrollment for each subject was decided by three same physicians. All subjects were informed
      that this procedure was a new technique, and that its effectiveness has not been confirmed,
      although an optimal outcome in patients with blastocyst transfer was obtained by using this
      technique for the same purpose (6). During the selection procedure, four eligible subjects
      opted not to participate when counseled about the uncertain of effectiveness, and other two
      subjects declined to randomize. Finally, 90 eligible patients were enrolled.

      Randomization First, ninety random numbers generated by means of computer were randomly
      divided into study group and control group. Then, all subjects in an enrolled date order were
      arranged according to the sequence of random numbers. So, ninety subjects were randomly
      divided into the study group and control group. The random allocation sequence was concealed
      in a closed and dark-colored envelope until the day of oocyte pick-up. Randomization occurred
      after patients agreed to participate in the study (Figure 1). All women of two groups
      underwent a conventional IVF or ICSI procedure, but in study group, the patients were added
      with a uterine cavity injection of day 2 embryo culture supernatant. The IVF or ICSI
      procedure was performed by same physicians and laboratory technicians. The treating sequence
      of subjects was as same as the enrolling sequence. The study was not blinded, because the
      patients as well as the clinicians were aware of the study content.

      IVF or ISCI procedure and injection technique of embryo culture supernatant Controlled
      ovarian hyperstimulation was performed using a variety of protocols, as previously published
      elsewhere (8). A down-regulation of the pituitary was realized during triptorelin
      (Triptorelin Acetate Injection, Ferring, Germany) administration S.C., 0.1 mg/d×7, started
      from day 21 of the previous cycles. Ovarian stimulation started from day 3 of stimulated
      cycle by using recombinant follicle stimulating hormone (rFSH, Gonal-F, Serono, Switzerland),
      225-300IU daily S.C.. The follicle development was monitored by transvaginal ultrasound scan
      (TVS). 250ug of HCG (Ovitrelle; Serono, Madrid) was administered by I.M. when one to two
      leading follicle(s) reached a mean diameter of 18 mm. In general, transvaginal oocyte
      retrieval was performed after 36 h of HCG injection.

      The media used for embryo culture was IVF-60. Briefly, after oocyte retrieval (day 0),
      cumulus-enclosed oocytes were incubated in IVF-60. Semen was prepared with a 45-90
      discontinuous gradient. The resulting pellet was washed in IVF-60. For normal IVF, 100,000 to
      150,000 sperm/100ul were used for each oocyte. The maturation stage of oocyte was assessed
      for IVF 15-18 hour after insemination (day 1). Cumulus and corona cells were removed using
      drawn pipettes. When ICSI was performed, oocytes were denuded with HYASE. Each mature oocyte
      was placed in a 6-ul droplet of GAMETE-20. The partner's sperm was placed in a 6-ul droplet
      of ICSI-100. All droplets were overlaid with OVOIL. ICSI was performed using a inverted
      microscope with RI micromanipulators (INTEGRA Micromanipulation System, Research Instruments,
      UK). The CO2 incubators (Forma, USA) with 6% CO2 gas were used for embryo incubation. An
      embryo scoring system based on cleavage rate and morphologic features was used for the
      evaluation of embryo quality, as previously described elsewhere (9). Briefly, grade 1 is the
      best score and grade 5 is the worst. For example, a grade 1 embryo has blastomeres of equal
      size with no cytoplasmic fragmentation. A grade 2 embryo has symmetric blastomeres, but a
      small degree of cytoplasmic fragmentation involving ≤10% of the embryo's surface. Grades 3
      through 5 demonstrate progressive embryo cytoplasmic fragmentation and variable cell
      symmetry.

      On the day 2, the embryos were translated from the old dish to a new dish with fresh media
      and cultured continuously. In study group, the embryo culture supernatant at the old dish
      were aspirated by using embryo transfer catheter (COOK-5000, Australia) and injected into the
      patient's uterine cavity. ET was performed on the third day of embryo culture. Two to three
      embryos were selected to transfer for each woman. The number of embryos selected for transfer
      was based on morphologic criteria and some additional factors, such as duration and cause of
      infertility and age of the patient. Pregnancy was determined by carry out a urine pregnancy
      test after 14 days of ET. The embryo implantation, the number of gestational sacs, and embryo
      heart pulsation were determined by TVS in the seventh week of pregnancy. Except the day 2
      embryo culture supernatant injection, all of the above-mentioned clinical and laboratory
      procedures were identical between the study group and control group.

      Statistical analysis Baseline characteristics of the patients were expressed as mean
      ±standard deviation or frequencies and proportion (%). The pregnancy and implantation rates
      were calculated based on the cases of pregnancy and number of gestational sacs. A Chi-Square
      Test was used to compare the implantation and conception proportions between two groups. The
      Statistics Package for Social Science (SPSS 13.0; SPSS Inc.) was used for statistical
      analyses. Statistical significance was set at P<0.05.
    

Intervention name(s): uterine cavity injection of day 2 embryo culture supernatant
Intervention type(s): Procedure
Intervention description(s): On the day 2, the embryos were translated from the old dish to a new dish with fresh media and cultured continuously. In study group, the embryo culture supernatant at the old dish were aspirated by using embryo transfer catheter (COOK-5000, Australia) and injected into the patient's uterine cavity. But in the control group, this intervention was not done. Conventional ET was performed on the third day of embryo culture for both groups.
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Single (Participant)
Primary purpose: Treatment
Keywords: Embryo culture supernatant; injection; embryo transfer; embryo implantation rate