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403 caso clínico Live birth after transfer of vitrified-warmed embryo - Z . Z hang et al Assisted reproductive technology (ART) has been advancing rapidly, and patients with clinically difficult characteristics can be successfully treated. Unfertilized oocyte cryopreservation, one of the advancing techniques of ART, has been established. The survival rate after thawing still needed to be improved. Recently, the freezing method has been changing from slow cooling cryopreservation to ultra rapid cryopreservation, namely, vitrification. This technique assures a high survival rate1,2 and is being used for cryopreservation of oocytes and embryos as a clinical application more and more. The fertility of survivors who underwent cancer treatments such as chemotherapy, radiotherapy and bone marrow transplantation has been impaired by the treatments. Thus, it is imperative to reserve these cancer survivors’ fertility before cancer treatments by cryopreserving their ovaries, oocytes or embryos3,4. Oocyte cryopreservation, however, may also be appropriate in circumstances where embryo freezing is not possible e.g in cases of malignant disease in young women where the imminent cytotoxic therapy is likely to inhibit subsequent oocyte development and where there is no male partner or in cases of routine in vitro fertilization (IVF) when no sperm are retrieved. The efficiency and success of subsequent use of frozen oocytes in such cases will, inevitably, be dependent on factors including the ability to select embryos for transfer. Patients with oligozoospermia may benefit from intracytoplasmic sperm injection (ICSI) treatment. However, some of infertile couples face poor results after ICSI and subsequently use donor sperm. In the present study, we report the healthy outcome of two girls who were born after transfer of two vitrified-warmed embryos produced by ICSI using vitrified-warmed oocytes and frozen-thawed donor sperms. Case presentation The patient was a 27-year-old woman. Her husband had been diagnosed with severe oligoasthenspermia. Ovarian stimulation was achieved by the daily administration of recombinant FSH (150 IU/day) following pituitary desensitisation with GnRH agonist. Meiotic maturation of preovulatory oocytes was induced by administration of human chorionic gonadotrophin (hCG; 6,000 IU) and cumulus-oocyte complexes were retrieved transvaginally 36 h later under ultrasound guidance. Twenty-five oocyte-cumulus complexes were identified in the aspirated follicular fluid. About ten inactive sperms were found in her husband semen. Multiple site open testicular biopsy was performed on her partner on the day of oocyte collection but no sperm was retrieved. The couple opted to select half of oocytes to be fertilized and have the twelve mature oocytes cryopreserved pending a decision on future treatment. Following digestion of cumulus-oocyte complexes using hyaluronidase (10  IU/ml), 23 of the 25 oocytes were confirmed as being at the metaphase II stage of meiosis and 11 mature oocytes were used to intracytoplasmic sperm injection (ICSI) and other 12 of them were prepared for cryopreservation. Other oocytes were frozen 4 h after follicle aspiration and were stored in 2013 and remained in storage for five months prior to thawing. Two had 2 visible pronuclei at 17 h post insemination (hpi) after intracytoplasmic sperm injection by using the only about ten inactive sperms from her husband. On day-3 post-ICSI, the embryos in culture included two 6-cell embryos with < 20% fragmentation and were transferred. Pregnancy was not achieved after 14 days of embryos’ transfer. Straws containing cryopreserved oocytes were thawed rapidly. All 12 cryopreserved ooctyes were thawed, and 12 oocytes survived the thaw process. Cryopreserved donor sperm was prepared by density gradient centrifugation and intracytoplasmic sperm injection. Post injection oocytes were cultured in Quinn’s Advantage Fertilisation medium for 17 h and assessed for the presence of pronuclei before being transferred to Quinn’s Advantage Cleavage medium. Twelve oocytes underwent ICSI and 9 were successfully fertilized with 2 visible pronuclei. Timing of syngamy was assessed at 23 hpi and embryo development was assessed at 41 hpi prior to selection for cryopreservation. Consequently, on December 26th, 2013, two vitrified warmed cleavage embryos (Gardner’s criteria, 6 II and 6 II) were transferred to the patient, and two gestational sacs with fetal heart movements were observed by ultrasonography at eight weeks of gestation. Finally, this resulted in a birth of two normal healthy female, weighing 4,700  g and 4,000 g at 35 weeks of gestation by cesarean delivery on October 25th, 2014. The twins were all healthy after two years. Rev Med Chile 2017; 145: 402-405


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