1 IVF Units, Carmel Hospital, Rambam Medical Center, TECHNION and The Rappaport Faculty of Medicine, Haifa and 2 Department of Obstetrics and Gynecology, The Chaim Sheba Medical Center, Tel Hashomer, and Sackler School of Medicine, Tel Aviv University, Israel
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Abstract |
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Key words: embryo quality/implantation rate/in-vitro fertilization/pregnancy rates/zona hardening
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Introduction |
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The present study was conducted prospectively to evaluate the effect of a short exposure of oocytes to spermatozoa. The fertilization rate, embryo quality, implantation and pregnancy rates were noted. Oocytes collected were exposed to spermatozoa for either 1 h or the standard 1624 h incubation periods. Recent studies (Dirnfeld et al., 1993; Gianaroli et al., 1996a
) reported that the exposure of human oocytes to spermatozoa for several hours resulted in higher fertilization rates, and achieved better quality embryos than those with the use of longer overnight gamete co-incubation.
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Materials and methods |
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Patient selection and characteristics
Normo-ovulatory patients aged 23
41 years were enrolled in the study. Uterine morphology and endometrial line were normal in all patients (assessed by hysterosalpingography and ultrasound). Among the 158 patients selected, 107 were diagnosed as tubal factor infertility, and 23 as unexplained infertility. A further 28 patients were classified as mild oligoterato-azoospermia (OTA) by the WHO criteria (WHO, 1992). Very poor responders, patients with polycystic ovary syndrome and men with severe oligozoospermia were excluded from the study.
Protocol of ovarian stimulation and oocyte retrieval
Down-regulation using gonadotrophin-releasing hormone analogue (GnRHa) was performed in all patients, using buserelin (Suprefact®) nasal spray (Hoechst, Frankfurt, Germany) 1000 µg/day continued up to human chorionic gonadotrophin (HCG) administration. When laboratory testing indicated pituitary suppression (oestradiol <40 pg/ml), three ampoules per day of human menopausal gonadotrophin (HMG) were administered, until three or more follicles of >18 mm mean diameter were present on transvaginal ultrasound (TVS). Follicular monitoring by serum oestradiol, luteinizing hormone (LH), progesterone and serial TVS scan were performed as previously described (Dirnfeld et al., 1993). Patients then received 5000 IU of HCG i.m. 3536 h before the scheduled TVS-guided follicular aspiration.
Insemination and fertilization
Oocytes were scored for maturity at recovery and incubated for 45 h before fertilization in HTF medium, with 10% synthetic serum substitute (SSS; Irvine Scientific, Santa Ana, CA, USA). Sperm recovery was carried out by the discontinuous mini-Percoll gradient technique after an initial sperm analysis. Fertilization was performed using 2050 x103 motile spermatozoa per oocyte, in a 5 µl (total volume) oil overlay. In group I (short exposure) 732 oocytes were withdrawn from the insemination medium after exposure to spermatozoa for 1 h. These oocytes were rinsed gently and further cultured in fresh HTF-SSS medium. In group II (standard exposure) 822 oocytes were left to incubate with spermatozoa for the standard 1624 h. Observation of 2PN was performed in both groups 1620 h after the exposure of oocytes to spermatozoa. Cleavage was assessed after 4850 h and embryo quality was evaluated using the modified criteria of Cummins and Breen (1986). Up to four embryos were selected for transfer into the uterine cavity. Luteal support was performed using progesterone in oil, 100 mg/day i.m. Statistical data analyses were conducted using 2 analysis and Student's t-test.
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Results |
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Discussion |
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Although fertilization and cleavage rates were similar in short (1 h) and standard (1620 h) exposure intervals of oocytes to spermatozoa, the implantation and pregnancy rates were seen to be higher in the short exposure group. This could be attributed to the improved embryo quality observed after short exposure. Poor embryo quality and zona hardening are major obstacles to successful implantation. We suggest that short exposure of the oocyte to spermatozoa may have a favourable effect on implantation rates by improving embryo quality. This was also observed by Gianaroli (1996a, b) and Quinn et al. (1998). The overnight exposure of oocytes to large numbers of spermatozoa led to inferior embryo quality, which may have been due to suboptimal culture conditions, overloaded by the excessive amounts of ROS and other products of metabolites. Reactive oxygen species was also emphasized by others (Mazzilli et al., 1994; Dumoulin, et al., 1998). Moreover, ROS generated by leukocytes in the activated state may affect mature spermatozoa by leading to loss of motility (Mazzilli et al., 1994
). However, in contrast to our observations, The concept that short spermatozoaoocyte coincubation times improve embryo quality implantation or pregnancy rate was not supported (Free et al., 1998
). Differences in sperm concentration and total numbers of spermatozoa used for insemination, duration of oocyte and spermatozoa coincubation and differences in culture media used may explain these differing results.
It is our opinion that shortening of eggspermatozoa exposure may improve embryo morphology by preventing the negative effect caused by oxygen free radicals produced by the spermatozoa during prolonged exposure. It may be assumed that long exposure of the oocyte to spermatozoa also contributes to the zona hardening effect, through the release of zona hardening factors, such as tissue type plasminogen activators (Wasserman, 1988).
Acceleration of zona hardening has been reported in several situations, including unfertilized oocytes, in-vitro conditions and especially in elderly women (Cohen et al., 1992). Assisted hatching of the cleaved embryo is one of the techniques which were suggested to improve implantation rates in this group (Cohen et al., 1992
). Short exposure could prove to be a good alternative method to other techniques, such as assisted hatching. The procedure, which is physiological in nature, and cost effective, does not require invasive techniques such as micromanipulation, which may pose certain risks to the embryo.
In summary, our prospective, randomized study confirms previous results, and shows that long exposure of the oocyte to spermatozoa in vitro is not advantageous, and may be inferior to short exposure, as shown by the observed increase in pregnancy rate after short exposure. Moreover, short exposure may prove to have a favourable effect on embryo quality and implantation rates. Short exposure of the egg to spermatozoa in vitro may provide a good alternative to other assisted techniques.
Further study is required to elaborate on and understand the mechanism of eggsperm interactions in relation to their duration of exposure.
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Notes |
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References |
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Cummins, J.M. and Breen, T.M. (1986) Embryo gradings. J. In vitro Fert. Embryo. Transf., 5, 284295.
Dirnfeld, M., Goldman, S., Koifman, M. et al. (1993) Very short exposure of oocytes to normal sperm in vitro improves implantation rates: a prospective randomized study (Abstract 0154) In Proceedings of the 51st Annual Meeting of the American Society for Reproductive Medicine, 1995, American Society for Reproductive Medicine, Seattle, S76.
Dumoulin, J.C.M., Bras, M., Land, J.A. et al. (1992) Effect of the number of inseminated spermatozoa on subsequent human and mouse embryonic development in vitro. Hum. Reprod., 7, 10101013.[Abstract]
Free, D.A., Merryman, D.C., Stringfellow, S.E. et al. (1998) Limited oocyte-sperm co-incubation time does not improve embryo quality, implantation, or pregnancy rate in patients undergoing in vitro fertilization. Abstract 0263, IFFS `98, 16th World Congress on Fertility and Sterility, and 54th Annual Meeting of the American Society for Reproductive Medicine. Fertil. Steril. (Suppl.), 70, S98S99.
Gianaroli, L., Fiorentino, A., Magli, M.C. et al. (1996a) Prolonged spermoocyte exposure and high sperm concentration affect human embryo viability and pregnancy rate. Hum. Reprod., 11, 25072511.[Abstract]
Gianaroli, L., Magli, C.M., Ferrareti, A.P. et al. (1996b) Reducing the time of spermoocyte interaction in human in-vitro fertilization improves the implantation rate. Hum. Reprod., 11, 166171.[Abstract]
Mazzilli, F., Rossi, T., Marchesini, M. et al. (1994) Superoxide anion in human semen related to seminal parameters and clinical aspects. Fertil. Steril., 62, 862868.[ISI][Medline]
Quinn, P., Lydic, M.D., Ho, M. et al. (1998) Confirmation of the beneficial effects of brief coincubation of gametes in human in vitro fertilization. Fertil. Steril., 69, 399402.[ISI][Medline]
Wasserman, P.M. (1987) The biology of chemistry of fertilization. Science, 285, 553560.
Wasserman, P.M. (1988) Fertilization in mammals. Sci. Am., 259, 7884.[ISI][Medline]
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SemenCervical Mucus Interaction. Cambridge University Press, Cambridge.
Submitted on March 26, 1999; accepted on June 28, 1999.