High fertilization and implantation rates after intracytoplasmic sperm injection

Human Reproduction High fertilization and implantation rates after intracytoplasmic sperm injection Andre C.Van Steirteghem 0 1 Zsolt Nagy 0 1 Hubert Joris 0 1 Jiaen Liu 0 1 Catherine Staessen 0 1 Johan Smitz 0 1 Arjoko Wisanto 0 1 Paul Devroey 0 1 0 Centre for Reproductive Medicine, Academisch Ziekenhuis VUB , Laarbeeklaan 101, B-1090 Brussels , Belgium 1 World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm -Cervical Mucus Interaction. 3rd edn. Cambridge University Press , Cambridge 'To whom correspondence should be addressed D o w Patients laon d The controlled comparison between the SUZI and ICSI ed procedures was carried out between 3 August 1992 and 4 rfom September 1992 on 11 treatment cycles, which were included th t in the recently submitted report on 300 consecutive treatment :// p cycles (Van Steirteghem et al., 1993). The 150 consecutive uh m treatment cycles by ICSI in 150 couples were carried out between rep 20 October 1992 and 12 January 1993. The inclusion criteria .xo f for ICSI were (i) total absence or < 5 % of normal fertilization rdo after standard IVF, (ii) < 500 000 progressively motile jruo spermatozoa in the whole ejaculate, or (iii) failed or sporadic lan s fertilization after SUZI. Previous treatments were carried out in .ro g our centre or in the centres which referred the couples to our /b y centre specifically to have ICSI. ug Patient counselling included information about the novelty of tse o this new procedure of assisted fertilization. The patients signed n M a consent form which included prenatal diagnosis by chorionic ra c villus sampling or amniocentesis as well as a prospective h2 follow-up of the children born after the ICSI procedure. ,22 The protocol was reviewed and approved by the ethical 150 committee of the Medical Campus of the Dutch-speaking Brussels Free University. Ovarian stimulation - Previously reported better fertilization rate after intracytoplasmic single sperm injection (ICSI) than after subzonal insemination of several spermatozoa was confirmed in a controlled comparison of the two procedures in 11 patients. Intracytoplasmic sperm injection was carried out in 150 consecutive treatment cycles of 150 infertile couples, who had failed to have fertilized oocytes after standard in-vitro fertilization (IVF) procedures or who were not accepted for IVF because not enough motile spermatozoa were present in the ejaculate. A single spermatozoon was injected into the ooplasm of 1409 metaphase II oocytes. Only 117 oocytes (8.3%) were damaged by the procedure and 830 oocytes (64.2% of the successfully injected oocytes) had two distinct pronuclei the morning after the injection procedure. The fertilization rate was not influenced by semen characteristics. After 24 h of further in-vitro culture, 71.2% of these oocytes developed into embryos, which were transferred or cryopreserved. Only 15 patients did not have embryos replaced. Three-quarters of the transfers were triple-embryo transfers. High pregnancy rates were noticed since 67 pregnancies were achieved, of which 53 were clinical, i.e. a total and clinical pregnancy rate of 44.7% and 35.3% per started cycle and 49.6% and 39.2% per embryo transfer. A total of 237 supernumerary embryos were cryopreserved in 71 treatment cycles. Key words: intracytoplasmic injection/in-vitro fertilization/malefactor infertility /oocyte/spermatozoon Intracytoplasmic sperm injection (ICSI) has recently been described as beneficial in alleviating infertility in couples who could not be helped by standard in-vitro fertilization (TVF) treatment or by subzonal insemination (SUZI) of the oocytes (Palermo et al., 1992, 1993; Van Steirteghem et al., 1993). Most of these infertile couples suffered from severe male-factor infertility and the number of motile spermatozoa in the ejaculate was sometimes too low for the couples to be accepted in an IVF programme. This report describes the results of a controlled comparison of me SUZI and ICSI procedures on sibling oocytes in 11 treatment cycles as well as die outcome of 150 consecutive treatment cycles of assisted fertilization by ICSI in couples who failed to fertilize after IVF or after SUZI or who could not be accepted for IVF treatment because of extremely impaired semen characteristics. Materials and methods Ovarian stimulation was carried out by a desensitizing protocol of the intranasally administered gonadotrophin-releasing hormone agonist (GnRHa) buserelin (Suprefact; Hoechst, Brussels, Belgium) in association with human menopausal gonadotrophins (HMG; Humegon; Organon, Oss, The Netherlands; or Pergonal; Serono, Brussels, Belgium) and human chorionic gonadotrophins (HCG; Pregnyl, Organon; Profasi, Serono). The details of die stimulation protocols have been described previously (Smitz etal., 1988). The supplementation of the luteal phase was started on the day after HCG administration and consisted of natural micronized progesterone 600 mg per day intravaginally in three divided doses (Utrogestan, Piette, Brussels; Smitz etal, 1992, 1993). Semen evaluation and preparation The evaluation of semen density and motility was carried out according to the recommendations of the World Health Organization (WHO, 1992). Strict criteria were used to evaluate sperm morphology (Kruger et al., 1986). A semen sample was considered to be normal when the following criteria were fulfilled: (i) sperm density 20 x 106/ml, (ii) progressive motility of 40%, and (iii) at least 14% of spermatozoa with normal morphology. Semen evaluation and preparation was done at least once prior to the treatment cycle in order to evaluate whether enough spermatozoa were present in the ejaculate to perform ICSI. The semen preparation consisted of treatment on a Percoll discontinuous gradient and sometimes of a further treatment with electroporation or metabolic stimulants pentoxifylline and 2-deoxyadenosine. Oocyte preparation for micro-injection Oocyte retrieval was done by vaginal ultrasound-guided puncture of the ovarian follicles, 36 h after HCG administration. At the end of the oocyte retrieval, the cumuluscorona cell complexes were inspected under the inverted microscope at x40 or x 100 magnification and classified as mature, slightly immature, slightly hypermature or immature (Khan et al., 1989). The cumuluscorona cell complexes were transferred into 5 ml Falcon tubes with Earle's medium; these tubes were gassed (5% O2, 5% CO2, 90% N2) prior to being closed tightly and then transported in a thermobox at 37C to the micro-injection laboratory, which is located elsewhere on the Medical Campus at a distance of ~500 m. The cells of the cumulus and corona radiata were removed by incubation for ~ 30 s in HEPES-buffered Earle's medium with 80IU hyaluronidase/ml (type VIE, sp. act. 320 IU/mg, Sigma Chemical Co., St Louis, MO, USA). The removal of the cumulus and corona cells was enhanced by aspiration of the complexes in and out of hand-d (...truncated)