Human Reproduction, Vol. 14, No. 10, 2562-2564,
October 1999
© 1999 European Society of Human Reproduction and Embryology
Shortened exposure of oocytes to spermatozoa improves in-vitro fertilization outcome: a prospective, randomized, controlled study
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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A prospective, randomized study of 158 patients undergoing in-vitro fertilization (IVF) and embryo transfer was conducted to evaluate whether a shortened exposure of oocytes to spermatozoa enhances oocyte development, and subsequently influences the IVF outcome. A comparison was made between conventional treatment time and shorter exposure of retrieved oocytes to spermatozoa. Fertilization and cleavage rates, embryo quality, implantation and pregnancy rates in the study group (short exposure) versus controls (standard IVF procedure) were evaluated. Fertilization (56 versus 61%) and cleavage rates (96 versus 92%) were similar in the two groups respectively. However, embryo quality was significantly higher in the study group (P < 0.05). Moreover, the pregnancy and implantation rates were significantly increased (42.4 versus 26% per embryo transfer, and 16 versus 10% respectively; P < 0.05). Our results demonstrated that shorter exposure of oocytes to spermatozoa is superior to the standard time in IVF and may have a favourable effect on implantation rates by improving embryo quality.
Key words: embryo quality/implantation rate/in-vitro fertilization/pregnancy rates/zona hardening
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Introduction |
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Several studies have demonstrated that in humans, spermatozoa–egg interaction and fertilization occurs within ~20 min post-coitus (Wasserman, 1987
, 1988). In standard in-vitro fertilization (IVF), spermatozoa are introduced as soon as oocyte maturation is observed. Oocytes are exposed to spermatozoa for an average of 16–20 h, at which time the two pronuclei (2PN) are usually inspected. Numerous progressively motile spermatozoa around the oocyte and multiple sperm tails attached to the unfertilized egg are frequently seen. It is well documented that reinsemination at this stage fails to improve IVF results in terms of fertilization, implantation and pregnancy rates (Cohen et al., 1992). This may be partially due to the refractory state of the zona pellucida after sperm penetration. Sperm–zona interaction leads to a series of events, which results in a block to polyspermy by the process of zona hardening (Wasserman, 1988). It is reasonable to assume that long exposure of the egg to spermatozoa may also accelerate zona hardening. The latter is essential for normal development of the embryo after fertilization, and may present a major obstacle to successful implantation after IVF. Moreover, prolonged exposure between gametes may lead to toxic effects associated with reactive oxygen species (ROS) released by the spermatozoa.
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 16–24 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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A total of 158 patients undergoing IVF procedures was selected after providing individual informed consent. Patients were randomized into two study groups using standard random number tables. In group I (n = 72), the oocytes were incubated with spermatozoa for 1 h, then cumulus cells were removed and placed in another well containing human tubal fluid (HTF) medium. In group II (n = 86) standard overnight spermatozoa–egg incubation was performed, and the spermatozoa were incubated with oocytes for 16–20 h.
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. 35–36 h before the scheduled TVS-guided follicular aspiration.
Insemination and fertilization
Oocytes were scored for maturity at recovery and incubated for 4–5 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 20–50 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 16–24 h. Observation of 2PN was performed in both groups 16–20 h after the exposure of oocytes to spermatozoa. Cleavage was assessed after 48–50 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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The mean patient age, indication for IVF, sperm concentration of the male partner and the number of retrieved oocytes were similar in the study and control groups, as shown in Table I
. Fertilization rates, cleavage rates and the number of embryos did not differ significantly between the study and control groups. However, the proportion of good quality (grade I) embryos (Table II) was significantly higher in the study group than in the controls (P < 0.05). Pregnancy rates per embryo transfer, defined as fetal sac with heart rate, and implantation rates were significantly higher in the study group, compared with the controls. In group 1, three patients had twins, compared to four in group 2. The results are shown in Table III.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Our results show that fertilization in vitro occurs within a brief period after exposure of the oocyte to spermatozoa which is in accordance with previous observations (Dirnfeld et al., 1993
).
Although fertilization and cleavage rates were similar in short (1 h) and standard (16–20 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 spermatozoa–oocyte 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 egg–spermatozoa 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 egg–sperm interactions in relation to their duration of exposure.
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Notes |
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3 To whom correspondence should be addressed at: IVF Unit, Department of Obstetrics and Gynecology, Carmel Medical Center, 7 Michal Street, 34362 Haifa, Israel
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References |
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Cohen, J., Alikani, M., Trowbridge, J. et al. (1992) Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum. Reprod., 7, 685–691.
Cummins, J.M. and Breen, T.M. (1986) Embryo gradings. J. In vitro Fert. Embryo. Transf., 5, 284–295.
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 0–154) 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, 1010–1013.
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 0–263, IFFS `98, 16th World Congress on Fertility and Sterility, and 54th Annual Meeting of the American Society for Reproductive Medicine. Fertil. Steril. (Suppl.), 70, S98–S99.
Gianaroli, L., Fiorentino, A., Magli, M.C. et al. (1996a) Prolonged sperm–oocyte exposure and high sperm concentration affect human embryo viability and pregnancy rate. Hum. Reprod., 11, 2507–2511.
Gianaroli, L., Magli, C.M., Ferrareti, A.P. et al. (1996b) Reducing the time of sperm–oocyte interaction in human in-vitro fertilization improves the implantation rate. Hum. Reprod., 11, 166–171.
Mazzilli, F., Rossi, T., Marchesini, M. et al. (1994) Superoxide anion in human semen related to seminal parameters and clinical aspects. Fertil. Steril., 62, 862–868.[Web of Science][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, 399–402.[Web of Science][Medline]
Wasserman, P.M. (1987) The biology of chemistry of fertilization. Science, 285, 553–560.
Wasserman, P.M. (1988) Fertilization in mammals. Sci. Am., 259, 78–84.[Web of Science][Medline]
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction. Cambridge University Press, Cambridge.
Submitted on March 26, 1999; accepted on June 28, 1999.
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