Produção Nacional
Revisado por pares
The optimal time for intracytoplasmic sperm injection in the human is from 37 to 41 hours after administration of human chorionic gonadotropin
2004; Elsevier BV; Volume: 82; Issue: 6 Linguagem: Inglês
10.1016/j.fertnstert.2004.09.002
ISSN1556-5653
AutoresDmitri Dozortsev, Péter Nagy, S. Abdelmassih, Flávio G. Oliveira, Amanda Brasil, V. Abdelmassih, Michael P. Diamond, Roger Abdelmassih,
Tópico(s)Assisted Reproductive Technology and Twin Pregnancy
ResumoFertilization rates increased continuously with the time elapsed after administration of hCG, reaching a peak of 84% when intracytoplasmic sperm injection (ICSI) was performed >41 hours after hCG administration. However, the highest implantation rate, 24%, was achieved when ICSI was performed 37–41 hours after hCG administration. Fertilization rates increased continuously with the time elapsed after administration of hCG, reaching a peak of 84% when intracytoplasmic sperm injection (ICSI) was performed >41 hours after hCG administration. However, the highest implantation rate, 24%, was achieved when ICSI was performed 37–41 hours after hCG administration. It is well known that oocytes retain their ability to become fertilized much longer than their capacity to develop into viable embryos (1Lundin K. Sjogren A. Hamberger L. Reinsemination of one-day-old oocytes by use of intracytoplasmic sperm injection.Fertil Steril. 1996; 66: 118-121PubMed Google Scholar, 2Yuzpe A.A. Liu Z. Fluker M.R. Rescue intracytoplasmic sperm injection (ICSI)—salvaging in vitro fertilization (IVF) cycles after total or near-total fertilization failure.Fertil Steril. 2000; 73: 1115-1119Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 3Tucker M.J. Morton P.C. Wright G. Ingargiola P.E. Jones A.E. Sweitzer C.L. Factors affecting success with intracytoplasmic sperm injection.Reprod Fertil Dev. 1995; 7: 229-236Crossref PubMed Scopus (27) Google Scholar, 4Fahmy N.W. Benoit J. Bissonnette F. Duchesne C. Girard Y. Sullivan R. Impact of a second insemination on the results of an in vitro fertilization-embryo transfer (IVF-ET) program.J In Vitro Fert Embryo Transf. 1991; 8: 80-83Crossref PubMed Scopus (11) Google Scholar, 5Morton P.C. Yoder C.S. Tucker M.J. Wright G. Brockman W.D. Kort H.I. Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes after complete conventional fertilization failure.Fertil Steril. 1997; 68: 488-491Abstract Full Text PDF PubMed Scopus (78) Google Scholar). For instance, oocytes inseminated 55 hours or more after hCG administration fertilize only at a slightly lower rate than those inseminated approximately 40 hours after hCG administration (1Lundin K. Sjogren A. Hamberger L. Reinsemination of one-day-old oocytes by use of intracytoplasmic sperm injection.Fertil Steril. 1996; 66: 118-121PubMed Google Scholar, 2Yuzpe A.A. Liu Z. Fluker M.R. Rescue intracytoplasmic sperm injection (ICSI)—salvaging in vitro fertilization (IVF) cycles after total or near-total fertilization failure.Fertil Steril. 2000; 73: 1115-1119Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 3Tucker M.J. Morton P.C. Wright G. Ingargiola P.E. Jones A.E. Sweitzer C.L. Factors affecting success with intracytoplasmic sperm injection.Reprod Fertil Dev. 1995; 7: 229-236Crossref PubMed Scopus (27) Google Scholar, 4Fahmy N.W. Benoit J. Bissonnette F. Duchesne C. Girard Y. Sullivan R. Impact of a second insemination on the results of an in vitro fertilization-embryo transfer (IVF-ET) program.J In Vitro Fert Embryo Transf. 1991; 8: 80-83Crossref PubMed Scopus (11) Google Scholar, 5Morton P.C. Yoder C.S. Tucker M.J. Wright G. Brockman W.D. Kort H.I. Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes after complete conventional fertilization failure.Fertil Steril. 1997; 68: 488-491Abstract Full Text PDF PubMed Scopus (78) Google Scholar). However, the implantation rate of the resulting embryos declines to nearly zero over the same period (1Lundin K. Sjogren A. Hamberger L. Reinsemination of one-day-old oocytes by use of intracytoplasmic sperm injection.Fertil Steril. 1996; 66: 118-121PubMed Google Scholar, 2Yuzpe A.A. Liu Z. Fluker M.R. Rescue intracytoplasmic sperm injection (ICSI)—salvaging in vitro fertilization (IVF) cycles after total or near-total fertilization failure.Fertil Steril. 2000; 73: 1115-1119Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 3Tucker M.J. Morton P.C. Wright G. Ingargiola P.E. Jones A.E. Sweitzer C.L. Factors affecting success with intracytoplasmic sperm injection.Reprod Fertil Dev. 1995; 7: 229-236Crossref PubMed Scopus (27) Google Scholar, 4Fahmy N.W. Benoit J. Bissonnette F. Duchesne C. Girard Y. Sullivan R. Impact of a second insemination on the results of an in vitro fertilization-embryo transfer (IVF-ET) program.J In Vitro Fert Embryo Transf. 1991; 8: 80-83Crossref PubMed Scopus (11) Google Scholar, 5Morton P.C. Yoder C.S. Tucker M.J. Wright G. Brockman W.D. Kort H.I. Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes after complete conventional fertilization failure.Fertil Steril. 1997; 68: 488-491Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Thus, the timing of insemination is apparently one of the important factors determining embryo viability. Although the developmental capacity of the oocyte declines significantly by 55 hours after hCG administration, according to several reports intracytoplasmic sperm injection (ICSI) performed at any time between 35 and 45 hours after hCG administration yields similar pregnancy rates, suggesting that the optimal insemination window is at least 10 hours (6Rienzi L. Ubaldi F. Anniballo R. Cerulo G. Greco E. Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection.Hum Reprod. 1998; 13: 1014-1019Crossref PubMed Scopus (120) Google Scholar, 7Jacobs M. Stolwijk A.M. Wetzels A.M. The effect of insemination/injection time on the results of IVF and ICSI.Hum Reprod. 2001; 16: 1708-1713Crossref PubMed Scopus (30) Google Scholar, 8Andrews M.M. Fishel S.B. Rowe P.H. Berry J.A. Lisi F. Rinaldi L. Analysis of intracytoplasmic sperm injection procedures related to delayed insemination and ejaculated, epididymal and testicular spermatozoa.Reprod Biomed Online. 2001; 2: 89-97Abstract Full Text PDF PubMed Scopus (10) Google Scholar, 9Isiklar A. Mercan R. Balaban B. Alatas C. Aksoy S. Urman B. Impact of oocyte pre-incubation time on fertilization, embryo quality and pregnancy rate after intracytoplasmic sperm injection.Reprod Biomed Online. 2004; 8: 682-686Abstract Full Text PDF PubMed Scopus (31) Google Scholar). Yanagida et al. (10Yanagida K. Yazawa H. Katayose H. Suzuki K. Hoshi K. Sato A. Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection.Hum Reprod. 1998; 13: 2223-2226Crossref PubMed Scopus (49) Google Scholar) demonstrated a significant decline in pregnancy rates when sperm injection was performed >44 hours after hCG administration. However, the rather low survival, fertilization, cleavage, and pregnancy rates reported in the study (10Yanagida K. Yazawa H. Katayose H. Suzuki K. Hoshi K. Sato A. Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection.Hum Reprod. 1998; 13: 2223-2226Crossref PubMed Scopus (49) Google Scholar) make the data difficult to interpret. This estimated 10-hour period seems rather long compared with the approximately 2-hour optimal insemination window in mice (11Edgar D.H. Whalley K.M. Mills J.A. Preimplantation development following in vitro fertilization of mouse oocytes: effect of timing of superovulation and preincubation in vitro.J In Vitro Fert Embryo Transfer. 1987; 4: 111-115Crossref PubMed Scopus (9) Google Scholar). This large window could be a real phenomenon, but it cannot be excluded that some factors obscure the true impact of insemination timing in the human. For example, in the very first study on the impact of timing (12Nagy Z.P. Joris H. Liu J. Staessen C. Devroey P. Van Steirteghem A.C. Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes.Hum Reprod. 1993; 8: 2180-2184Crossref PubMed Scopus (90) Google Scholar), investigators used embryo quality and not implantation as an end point outcome measure. In another study, the largest group of cycles examined with regard to the impact of timing was composed of conventional IVF (7Jacobs M. Stolwijk A.M. Wetzels A.M. The effect of insemination/injection time on the results of IVF and ICSI.Hum Reprod. 2001; 16: 1708-1713Crossref PubMed Scopus (30) Google Scholar). The rationale of combining the results obtained with conventional IVF and ICSI could be questioned, because the mechanisms of fertilization during ICSI and conventional insemination are not the same (13Dozortsev D. De Sutter P. Dhont M. Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection.Hum Reprod. 1994; 9: 2139-2144PubMed Google Scholar, 14Dozortsev D. Rybouchkin A. De Sutter P. Dhont M. Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for the sperm nucleus decondensation.Hum Reprod. 1995; 10: 2960-2965PubMed Google Scholar). In fact, it is possible that the only similarity is a sperm-derived factor (15Dozortsev D. Rybouchkin A. De Sutter P. Qian C. Dhont M. Human oocyte activation following intracytoplasmic injection: the role of the sperm cell.Hum Reprod. 1995; 10: 403-407PubMed Google Scholar), likely to be phospholipase C ζ (16Saunders C.M. Larman M.G. Parrington J. Cox L.J. Royse J. Blayney L.M. et al.PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development.Development. 2002; 129: 3533-3544Crossref PubMed Google Scholar), that triggers oocyte activation in both cases. Whereas oocyte activation timing after ICSI is relatively well defined, and activation has been shown to begin within 30 minutes after sperm injection (17Dozortsev D. Qian C. Ermilov A. Rybouchkin A. De Sutter P. Dhont M. Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction.Hum Reprod. 1997; 12: 2792-2796Crossref PubMed Scopus (52) Google Scholar, 18Dozortsev D. De Sutter P. Rybouchkin A. Dhont M. Timing of sperm and oocyte nuclear progression after intracytoplamic sperm injection.Hum Reprod. 1995; 10: 3012-3017PubMed Google Scholar), oocyte activation after conventional insemination cannot be estimated as precisely, owing to variation in the properties of the sperm sample, corona cells, zona pellucida, and other factors. In addition, existing studies of the impact of the insemination timing are typically extended over a period of several years (7Jacobs M. Stolwijk A.M. Wetzels A.M. The effect of insemination/injection time on the results of IVF and ICSI.Hum Reprod. 2001; 16: 1708-1713Crossref PubMed Scopus (30) Google Scholar, 8Andrews M.M. Fishel S.B. Rowe P.H. Berry J.A. Lisi F. Rinaldi L. Analysis of intracytoplasmic sperm injection procedures related to delayed insemination and ejaculated, epididymal and testicular spermatozoa.Reprod Biomed Online. 2001; 2: 89-97Abstract Full Text PDF PubMed Scopus (10) Google Scholar, 9Isiklar A. Mercan R. Balaban B. Alatas C. Aksoy S. Urman B. Impact of oocyte pre-incubation time on fertilization, embryo quality and pregnancy rate after intracytoplasmic sperm injection.Reprod Biomed Online. 2004; 8: 682-686Abstract Full Text PDF PubMed Scopus (31) Google Scholar, 10Yanagida K. Yazawa H. Katayose H. Suzuki K. Hoshi K. Sato A. Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection.Hum Reprod. 1998; 13: 2223-2226Crossref PubMed Scopus (49) Google Scholar), allowing some other factors, such as experience and procedural and personnel changes, to impact the outcome. Finally, the timing itself is usually vaguely defined (7Jacobs M. Stolwijk A.M. Wetzels A.M. The effect of insemination/injection time on the results of IVF and ICSI.Hum Reprod. 2001; 16: 1708-1713Crossref PubMed Scopus (30) Google Scholar, 8Andrews M.M. Fishel S.B. Rowe P.H. Berry J.A. Lisi F. Rinaldi L. Analysis of intracytoplasmic sperm injection procedures related to delayed insemination and ejaculated, epididymal and testicular spermatozoa.Reprod Biomed Online. 2001; 2: 89-97Abstract Full Text PDF PubMed Scopus (10) Google Scholar, 9Isiklar A. Mercan R. Balaban B. Alatas C. Aksoy S. Urman B. Impact of oocyte pre-incubation time on fertilization, embryo quality and pregnancy rate after intracytoplasmic sperm injection.Reprod Biomed Online. 2004; 8: 682-686Abstract Full Text PDF PubMed Scopus (31) Google Scholar, 10Yanagida K. Yazawa H. Katayose H. Suzuki K. Hoshi K. Sato A. Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection.Hum Reprod. 1998; 13: 2223-2226Crossref PubMed Scopus (49) Google Scholar). The most recent report (9Isiklar A. Mercan R. Balaban B. Alatas C. Aksoy S. Urman B. Impact of oocyte pre-incubation time on fertilization, embryo quality and pregnancy rate after intracytoplasmic sperm injection.Reprod Biomed Online. 2004; 8: 682-686Abstract Full Text PDF PubMed Scopus (31) Google Scholar) on the effect of insemination timing has demonstrated that sperm injected immediately after oocyte aspiration (approximately 36 hours after hCG administration) results in a lower implantation rate than after preincubation for at least 2 hours. However, the most significant practical question, the duration of the optimal insemination window, remained unanswered. Given the potential importance of insemination timing as one of the few variables that can be controlled, we believed that the question of insemination window merited further investigation. Therefore, the present work was undertaken to re-examine the issue of optimal insemination window, taking advantage of a large number of well-timed ICSI cycles performed over a short period. This project was approved by the institutional review board of the Clinica e Centro de Pesquisa em Reproducao Humana Roger Abdelmassih. No consent was obtained from the patients because the research involved only retrospective chart review, and no individual patient was identified in the manuscript by name or in any other manner. The timing was based on the time elapsed between the time of hCG injection and beginning of ICSI. Intracytoplasmic sperm injection was performed by two embryologists, one assisting by handling oocytes and the other performing injections. The timing was recorded immediately before injection. Injection took 20 minutes on average. A case was assigned to the timing group on the basis of the time when injection was initiated. The timing of case assignment was random, determined only by the work load on a given day. A total of 651 ICSI treatment cycles performed in Clinica Andrologia, São Paulo, Brazil between August 2002 and January 2003 were reviewed for this study. Cases of testicular sperm extraction and testicular sperm aspiration were excluded because they would usually take longer to perform. Semen samples were collected by masturbation and allowed to liquefy for approximately 20 minutes at 37°C before analysis. The analysis and treatment of semen samples have been described previously (18Dozortsev D. De Sutter P. Rybouchkin A. Dhont M. Timing of sperm and oocyte nuclear progression after intracytoplamic sperm injection.Hum Reprod. 1995; 10: 3012-3017PubMed Google Scholar). Patients were down-regulated with a GnRH agonist (leuprolide acetate 1 mg daily) (Reliser; Serono, São Paulo, Brazil) for an average of 14 days. The dose of GnRH agonist was reduced to 0.5 mg daily when the stimulation started. Controlled ovarian stimulation was carried out by administration of recombinant (r)FSH (Gonal-F; Serono) (300 IU/day) for the first 5 days of stimulation. Oocytes were recovered by ultrasound-guided follicular aspiration at 35 hours after hCG administration. Removal of the cumulus and corona cells was performed in a 50-μL droplet of GAMETE 100 medium (Vitrolife Inc., Englewood, CO) under oil to maintain the temperature and to limit evaporation of the medium. Intracytoplasmic sperm injection was performed as described elsewhere (19Palermo G. Joris H. Devroey P. Van Steirteghem A.C. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte.Lancet. 1992; 340: 17-18Abstract PubMed Scopus (2854) Google Scholar, 20Abdelmassih R. Sollia S. Moretto M. Acosta A.A. Female age is an important parameter to predict treatment outcome in intracytoplasmic sperm injection.Fertil Steril. 1996; 65: 573-577PubMed Google Scholar). Briefly, oocytes were placed in drops of N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered human tubal fluid (HTF) media. A washed aliquot of sperm cells was placed in a central drop containing HEPES-buffered HTF media with 5% polyvinylpyrrolidone. For ICSI sperm cells, sperm cells were immobilized by "touching tail" and picked up tail first by injection pipette and transferred into oocyte-containing drops. Sperm cells were injected at 3 o'clock relative to the polar body. After injection, oocytes were incubated in 1.0 mL of IVF 100 medium (Vitrolife) under oil in an atmosphere of 5% CO2 in air at 37°C. Oocytes were checked 17 to 18 hours after injection procedures. Normal fertilization was defined by the presence of two distinct pronuclei and two polar bodies. Cleavage of fertilized oocytes was assessed at 72 hours after injection; for each embryo, the number and size of blastomeres were recorded, as well as the percentage of fragmentation. Embryos were classified according to the following quality criteria on day 3: excellent quality (at least six cells and 20% fragmentation), grade C. For ET, an Edwards–Wallace 23-cm catheter (SIMS Portex, Hythe, Kent, United Kingdom) connected to an insulin syringe was used. The catheter was rinsed with transfer medium (IVF-50; Scandinavian IVF Science, Gothenburg, Sweden), then embryos were loaded and the catheter handed to the clinician, who inserted it through the endocervical canal under ultrasound guidance. Progesterone oral administration started on the day of the retrieval (800 mg daily Utrogestan; Besins Laboratories, Paris, France) and was continued until the assessment of pregnancy (12 days after transfer). Cephalexin was administered (500 mg every 6 hours for 5 days) starting on the day of retrieval. The β-hCG serum levels were assessed on day 12 after transfer. A clinical pregnancy was defined as the visualization of a heartbeat by transvaginal ultrasound at 6 weeks' gestation. One-way analysis of variance, χ2-test, and Fisher's exact test were applied wherever appropriate for statistical analysis. Results were considered significant at P<.05. Female patient age, number of oocytes aspirated per patient, number of oocytes injected per patient, number of embryos transferred, and the proportion of excellent embryos among embryos transferred were not statistically different between groups. Fertilization rates increased significantly as time after hCG injection elapsed. Pregnancy rates and clinical pregnancy rates peaked between 37 and 41 hours after hCG administration and declined when injection was performed later. Implantation and ongoing implantation rates reached the highest level when ICSI was performed between 37 and 39 hours after hCG administration. Miscarriage rates were similar between groups, with the exception of a trend toward higher miscarriages rates when ICSI was performed <37 hours after hCG administration. The lost fetal sacs rate was not statistically different between groups. The data are presented in Table 1.Table 1In vitro fertilization cycle outcome measures as a function of timing of injection.Hours after hCG injectionAge (y)No. of oocytes per patient (aspirated)No. of oocytes per patient (injected)Fertilization rateNo. of embryos transferredNo. of embryos "A"Pregnancy rateClinical pregnancy rateImplantation rateOngoing implantation rateLost sacs rateMiscarriage rate<3736.1 ± 5.710.5 ± 7.67.2 ± 4.0327/453 (72)3.3 ± 1.52.0 ± 1.522/65 (34)20/63 (32)31/206 (15)23/206 (11)8/31 (26)7/20 (35)37–3935.6 ± 5.711.4 ± 9.27.2 ± 3.71490/1900 (78)3.2 ± 1.42.1 ± 1.4131/281 (47)115/265 (43)206/861 (24)157/861 (18)49/206 (24)23/115 (20) P vs. <37 h.0005.0051.016839–4135.9 ± 5.812 ± 9.67.7 ± 4.01454/1777 (82)3.5 ± 1.52.2 ± 1.6114/240 (48)104/231 (45)180/807 (22)136/807 (17)44/180 (24)20/104 (19) P vs. <37 h<.0001.0212 P vs. 37–39 h.0104.0466>4136.7 ± 5.611 ± 8.97.3 ± 3.7548/655 (84)3.2 ± 1.52.5 ± 1.630/92 (33)28/90 (31)50/289 (17)38/289 (13)12/50 (24)6/28 (21) P vs. <37 h<.0001 P vs. 37–39 h.004.0212.0466.0219 P vs. 39–41 h.0185.0237Note: Data are presented as mean ± SD or n (%). "A" = excellent quality (at least six cells with <10% fragmentation). Open table in a new tab Note: Data are presented as mean ± SD or n (%). "A" = excellent quality (at least six cells with <10% fragmentation). Our results demonstrate that human oocytes have a relatively narrow optimal insemination time window (Table 1). We believe that the failure of previous studies to detect this phenomenon was mainly due to the long duration of the studies and vaguely defined timing. Our findings are new but not surprising. In mice, for instance, in vitro insemination delayed by just 2 hours results in significantly less development to the blastocyst stage (11Edgar D.H. Whalley K.M. Mills J.A. Preimplantation development following in vitro fertilization of mouse oocytes: effect of timing of superovulation and preincubation in vitro.J In Vitro Fert Embryo Transfer. 1987; 4: 111-115Crossref PubMed Scopus (9) Google Scholar). It has also been shown that human oocytes retain their ability to become fertilized with conventional insemination longer than their ability to develop into viable embryos (4Fahmy N.W. Benoit J. Bissonnette F. Duchesne C. Girard Y. Sullivan R. Impact of a second insemination on the results of an in vitro fertilization-embryo transfer (IVF-ET) program.J In Vitro Fert Embryo Transf. 1991; 8: 80-83Crossref PubMed Scopus (11) Google Scholar). Similarly, an initial optimism regarding "rescue ICSI" on day 1 (12Nagy Z.P. Joris H. Liu J. Staessen C. Devroey P. Van Steirteghem A.C. Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes.Hum Reprod. 1993; 8: 2180-2184Crossref PubMed Scopus (90) Google Scholar) has quickly disappeared because embryos conceived apparently failed to develop to term (2Yuzpe A.A. Liu Z. Fluker M.R. Rescue intracytoplasmic sperm injection (ICSI)—salvaging in vitro fertilization (IVF) cycles after total or near-total fertilization failure.Fertil Steril. 2000; 73: 1115-1119Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 5Morton P.C. Yoder C.S. Tucker M.J. Wright G. Brockman W.D. Kort H.I. Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes after complete conventional fertilization failure.Fertil Steril. 1997; 68: 488-491Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Our data show that the ability of human oocytes to develop into viable embryos increases with the interval between hCG administration and ICSI, reaching a peak between 37 and 39 hours after hCG administration and then gradually declining. Oocytes injected earlier than 37 hours after hCG administration fertilized at significantly lower rates. We attribute this to oocyte cytoplasmic immaturity at the time of injection. It has been well established by studies in other mammals that oocytes gradually develop their ability to become fertilized and that ooplasm maturity lags behind nuclear maturity (21Kubiak J.Z. Mouse oocytes gradually develop the capacity for activation during the metaphase II arrest.Dev Biol. 1989; 136: 537-545Crossref PubMed Scopus (189) Google Scholar). The first polar body, which is usually used as a marker of maturity, is extruded before an oocyte is able to become fertilized (21Kubiak J.Z. Mouse oocytes gradually develop the capacity for activation during the metaphase II arrest.Dev Biol. 1989; 136: 537-545Crossref PubMed Scopus (189) Google Scholar). If fertilization is attempted after the first polar body extrusion but before the ooplasm is completely matured, then the resumption of meiosis might fail or be incomplete, with chromosomes blocked at so-called metaphase III, without the second polar body being extruded and without pronuclei being formed (21Kubiak J.Z. Mouse oocytes gradually develop the capacity for activation during the metaphase II arrest.Dev Biol. 1989; 136: 537-545Crossref PubMed Scopus (189) Google Scholar). It is interesting to note that the optimal insemination window found in this study begins approximately at the time when ovulation would have taken place. In agreement with earlier data from others (7Jacobs M. Stolwijk A.M. Wetzels A.M. The effect of insemination/injection time on the results of IVF and ICSI.Hum Reprod. 2001; 16: 1708-1713Crossref PubMed Scopus (30) Google Scholar), we found increased miscarriage rates in patients with ICSI performed earlier than 37 hours after aspiration. Because most first-trimester miscarriages are due to chromosomal aberrations (22Strom C.M. Ginsberg N. Applebaum M. Bozorgi N. White M. Caffarelli M. et al.Analyses of 95 first-trimester spontaneous abortions by chorionic villus sampling and karyotype.J Assist Reprod Genet. 1992; 9: 458-461Crossref PubMed Scopus (72) Google Scholar), one could speculate that perhaps some type of spindle immaturity at the time of injection, resulting in chromosomal aberrations, might be responsible for this finding. However, the lost fetal sacs rates in this group were similar to those of other groups, suggesting that the seemingly higher miscarriages rate is due to the fewer number of multiple pregnancies: loss of one fetal sac in a singleton pregnancy results in a miscarriage, whereas loss of one sack in a multiple gestation (vanishing twins) does not usually lead to a miscarriage. In turn, the lower number of multiple pregnancies is apparently due to the significantly lower implantation rate in this group. Interestingly, the fertilization rates increased over time, with the highest fertilization rates achieved in the "oldest" group of oocytes, even as implantation rates declined. It is important to note that fertilization in our study was achieved with sperm injection, and therefore the difference in fertilization rates reflects only an oocyte's sensitivity to the sperm-associated oocyte-activating factor (15Dozortsev D. Rybouchkin A. De Sutter P. Qian C. Dhont M. Human oocyte activation following intracytoplasmic injection: the role of the sperm cell.Hum Reprod. 1995; 10: 403-407PubMed Google Scholar). Therefore, the increase in fertilization over time would not necessarily take place during natural fertilization or conventional IVF, when other factors (e.g., zona binding, sperm-oocytes membrane fusion) would make their contribution, perhaps lowering the fertilization rates as oocytes become older. It is worth mentioning that although ICSI allows synchronization of insemination and activation within a cohort of the oocytes (17Dozortsev D. Qian C. Ermilov A. Rybouchkin A. De Sutter P. Dhont M. Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction.Hum Reprod. 1997; 12: 2792-2796Crossref PubMed Scopus (52) Google Scholar), this does not lead to synchronization of the subsequent fertilization events, such as chromatids separation, anaphase and telophase of the second meiotic division, pronuclei formation, and subsequent integration of replicated chromosomes into the metaphase plate of the first mitotic division (18Dozortsev D. De Sutter P. Rybouchkin A. Dhont M. Timing of sperm and oocyte nuclear progression after intracytoplamic sperm injection.Hum Reprod. 1995; 10: 3012-3017PubMed Google Scholar). As a result, the timing of the second polar body extrusion and to a lesser extent the timing of the appearance of pronuclei can vary widely within a cohort of oocytes, whether they are inseminated with ICSI or a conventional technique (18Dozortsev D. De Sutter P. Rybouchkin A. Dhont M. Timing of sperm and oocyte nuclear progression after intracytoplamic sperm injection.Hum Reprod. 1995; 10: 3012-3017PubMed Google Scholar). This variation could be attributed to the difference between spermatozoa or oocytes within a cohort and requires further investigation. It might also be due to the minute variations in sperm injection techniques (14Dozortsev D. Rybouchkin A. De Sutter P. Dhont M. Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for the sperm nucleus decondensation.Hum Reprod. 1995; 10: 2960-2965PubMed Google Scholar). There is strong evidence of a correlation between lower implantation rates and higher miscarriage rates in women of advanced reproductive age (20Abdelmassih R. Sollia S. Moretto M. Acosta A.A. Female age is an important parameter to predict treatment outcome in intracytoplasmic sperm injection.Fertil Steril. 1996; 65: 573-577PubMed Google Scholar). Both are believed to be due to chromosomal aberrations. Therefore, it is noteworthy that in the current study there was no correlation between implantation and miscarriage rates. Furthermore, despite a significant difference in implantation rates, lost fetal sacs rates were nearly identical in all groups. Therefore, the apparent difference in implantation potential observed in this study might be due to something other than chromosomal factors. Perhaps it is due to the decline in metabolic activity of the oocyte. Indeed, earlier data implicate the differences in adenosine triphosphate levels and mitochondrial potential in embryo viability (23Van Blerkom J. Davis P.W. Lee J. ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer.Hum Reprod. 1995; 10: 415-424Crossref PubMed Scopus (529) Google Scholar). Even if the lower implantation rates achieved 41 hours after hCG administration are due to metabolic incompetence of the oocytes, the specific mechanisms are not likely to be the same. The lower implantation rates after the earlier injection might be due to metabolic immaturity, whereas lower implantation after later injection might be due to metabolic "postmaturity." In conclusion, the timing of ICSI seems to be more important than has been previously realized. The optimal timing for injection is 37–41 hours after hCG administration. This might be important not only for ICSI but also for other timed assisted reproduction techniques.
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