Centre for Reproductive Medicine, Academic Hospital, Dutch-speaking Brussels Free University, Laarbeeklaan 101, B-1090 Brussels, Belgium
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Abstract |
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Key words: asthenozoospermia/controlled study/fertilization failure/IVF versus ICSI/motility
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Introduction |
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It is clear that the severe male indications can be treated successfully only with ICSI. It is less clear whether a difference in efficiency between IVF and ICSI exists for the real borderline male cases that theoretically fulfil the criteria for conventional IVF. While teratozoospermia has been widely studied, the impact of borderline asthenozoospermia on fertilization and embryo quality after conventional IVF and ICSI is less clear. In most centres, a cut-off value for progressively motile spermatozoa after preparation is defined to determine the admittance of a couple to the conventional IVF programme. A minimum total number of rapid (type A, >25 µm/s) and slow (type B, 525 µm/s) progressively motile spermatozoa together is generally used (WHO, 1992); in our centre this number is 500 000 type A+B spermatozoa. The question arises as to whether the predominant presence of slow progressive type B spermatozoa in semen may be an indication for ICSI, or a contra-indication for conventional IVF.
The aim of the present prospective, controlled study was therefore to compare the efficiency of conventional IVF and ICSI in cases of asthenozoospermia, defined in this study as 5% type A motility in the fresh semen sample.
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Materials and methods |
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In total, 20 cycles (20 couples) were carried out. The mean patient age was 31.6 ± 3.4 years (range 2539) for the female partners and 35.4 ± 4.9 years (range 2745) for the male partners.
Ovarian stimulation
Female partners underwent ovarian stimulation using a gonadotrophin-releasing hormone analogue suppression protocol with buserelin (Suprefact nasal spray; Hoechst, Frankfurt, Germany) combined with human menopausal gonadotrophins (HMG). Oocyte-cumulus complexes were recovered by vaginal ultrasound-guided retrieval 36 h after administration of 10 000 IU of human chorionic gonadotrophin (HCG).
Semen evaluation and preparation
Fresh semen samples were provided by masturbation on the day of oocyte collection. After liquefaction, sperm concentration and motility were assessed according to the recommendations of the World Health Organization (1992). Sperm morphology was evaluated using the strict Kruger criteria (Kruger et al., 1986). Semen samples from all patients included in the study showed
5% rapid progressive motility. All semen samples were prepared by discontinuous density gradient centrifugation, initially based on Percoll (95%47.5%, Pharmacia, Uppsala, Sweden), but later replaced by Pure Sperm (90%45%, Nidacon Laboratories, Göteborg, Sweden). A 1 ml fraction of fresh semen was placed on a gradient consisting of 1 ml of the bottom fraction and 1 ml of the top fraction. Multiple gradients were made whenever necessary. After centrifugation at 300 g for 20 min, 200400 µl of the bottom fraction were aspirated and washed twice before evaluation. After concentration and motility had been assessed, this fraction was either diluted or concentrated depending on the number of progressively motile (type A+B) spermatozoa per microlitre.
Oocyte retrieval and preparation
Oocyte retrieval was carried out by ultrasound-guided transvaginal puncture of the follicles 36 h after 10 000 IU HCG administration. The first cumulusoocyte complex was allocated either to ICSI or to conventional IVF according to a randomization table. The subsequent cumulusoocyte complexes were further alternately allocated to ICSI or IVF; those used for IVF were individually cultured in 25 µl droplets of B2 medium under paraffin oil.
In conventional IVF, oocytes were inseminated with 5000 progressively motile spermatozoa (type A+B) at least 2 h after ovum retrieval. The volume to be added ranged between 1 and 5 µl. If after concentrating the final sperm fraction the calculated volume providing 5000 motile cells exceeded 5 µl, the excess medium was first removed from the microdroplet before the sperm volume was added. The cumulusoocyte complexes allocated to ICSI were enzymatically denuded from their cumulus cells by exposure to a solution of 10 IU/ml hyaluronidase (Type VIII, Sigma Chemical Company, St Louis, MO, USA) followed by pipetting. Only the mature, metaphase-II oocytes were injected. Care was taken to perform the insemination of the IVF oocytes and the injection of the ICSI oocytes at the same hour of the day (around 14:00). The ICSI procedure was carried out as previously described (Van Steirteghem et al., 1995).
Fertilization, embryo quality and embryo transfer
Assessment of fertilization was carried out about 18 h after oocyte insemination and injection. Oocytes with two distinct pronuclei (2PN) together with two distinct or fragmented polar bodies were considered normally fertilized. For ICSI oocytes, the pronuclei had sometimes disappeared by that time, and these oocytes were considered normally fertilized when two polar bodies and normal cleavage were observed. Normal fertilization rate was always calculated as the number 2PN over the number of cumulusoocyte complexes, independent of the nuclear maturity, as well for IVF as for ICSI. At 4244 h post-insemination and post-injection, the morphological quality and developmental speed of the embryos were assessed. Embryos were classified into different categories according to the proportion of fragments: type A embryos without fragments, type B embryos with fragmentation up to 20%, type C embryos with fragmentation 20% and <50%, and type D embryos with
50% fragmentation. For each category, the percentage of embryos derived from 2PN fertilized oocytes was calculated. The normal cleaving embryos (type A, B and C) were also classified according to their speed of development, as 2-cell, 3- or 4-cell or 58 cell embryos. Embryos up to type C (<50% fragmentation) were replaced into the uterine cavity about 48 h after insemination or ICSI. Supernumerary good-quality embryos (type A or type B only) were frozen at day 2 or day 3.
The embryo transfer policy was the following: always the best-quality embryos were replaced, two (elective embryo transfer) or three depending on their morphological quality (Staessen et al., 1993), and preferably derived from the same treatment procedure (conventional IVF or ICSI). Where a similar high quality of the embryos allowed a choice between IVF and ICSI embryos, preference was given to the IVF embryos as the aim was to continue the treatment by the least complicated and the least expensive procedure (IVF), if pregnancy failed to occur. A rise in serum HCG on two consecutive occasions from day 11 onwards after embryo transfer indicated pregnancy. A clinical pregnancy was defined by the presence of a gestational sac at ultrasonography after approximately 7 weeks of pregnancy. Implantation rate was defined as the total number of fetal heart beats per total number of embryos replaced.
Parameters for evaluation
The following parameters were used to compare the efficiency of conventional IVF and ICSI: percentage 2PN fertilization, percentage 1 PN and 3 PN, morphological quality and rate of development of the embryos at 4244 h post-insemination and post-injection, number of embryos transferred on day 2, number of embryos frozen (on day 2 or 3) and total number of embryos transferred or frozen. The fertilization rate was always calculated as the number of 2PN oocytes compared to the number of retrieved cumulusoocyte complexes. As the origin of the embryos for transfer was not randomized, no conclusion could be drawn regarding the pregnancies.
Statistical analysis
The Wilcoxon signed-rank test for paired data was used to compare the above parameters on sibling oocytes. The MannWhitney U test was used to compare results between the group of patients with fertilization after conventional IVF and the group of patients without fertilization after conventional IVF. All tests were performed at the 5% level of significance.
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Results |
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Fertilization and embryo quality
The mean number of cumulusoocyte complexes per patient was 14.7, a mean of 7.4 oocytes to be treated by IVF and 7.3 oocytes to be treated by ICSI. Although only the mature, metaphase II oocytes were injected, percentages of 2PN, 1PN and 3PN per cycle have always been calculated as a proportion of the number of retrieved cumulusoocyte complexes (Table II
). This is considered to be the only way to carry out a valid comparison, as oocyte maturity on the day of oocyte retrieval is checked only for the ICSI oocytes and not for IVF oocytes. The fertilization rate after IVF (a mean of 22.9% per cycle) was far below the fertilization rate after ICSI (63.4%, P < 0.001). Most striking was the observation of complete fertilization failure by conventional IVF in 10 out of the 20 cycles (50%), while 2PN fertilization always ensued after ICSI. Patients with fertilization after IVF (n = 10) showed a reasonable fertilization rate of 45.7% after IVF, which was not significantly different from the 59.4% obtained after ICSI for the same 10 patients. Percentages of 1PN and
3 PN were similar for both treatment procedures.
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No difference between the two subpopulations was observed in the 2PN rate obtained after ICSI. The pregnancy rate was higher in the fert+ group, where the higher number of embryos available allowed a greater selection of the embryos for transfer.
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Discussion |
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Differences between IVF and ICSI are more pronounced in cases of male subfertility. For patients with borderline semen, ICSI has shown a better performance than conventional IVF in terms of fertilization rate and absence of fertilization failure (Aboulghar et al., 1995, 1996
; Calderon et al., 1995
). Aboulghar et al. reported a fertilization failure rate of 50% after IVF, which is very high with regard to the reported reasonable semen quality (Aboulghar et al., 1995
). Calderon and colleagues included a few patients with single, double, or triple sperm defects who should in fact be considered untreatable by conventional IVF (Calderon et al., 1995
). In each of his three defined categories of sperm defects, at least one patient had no progressively motile spermatozoa, which is a clear contra-indication for conventional IVF. Such remarks stress the variability in the interpretation of `borderline semen' quality between groups and argue for a critical analysis of the reported data. As regards teratozoospermia, a high risk of fertilization failure (11/18 patients) after IVF leading to extreme differences in fertilization rates between IVF and ICSI (76% for ICSI against 15% for IVF) has been reported (Payne et al., 1994
). This problem seems partly to be solved by the procedure of `high insemination concentration' for IVF (Ord et al., 1993
; Hall et al. 1995
), but remains in cases of severe teratozoospermia (<5% normal morphology) (Fishel et al., 1995
).
For asthenozoospermia, a controlled comparison of conventional IVF and ICSI has not yet been reported. In the present study, asthenozoospermia is defined as a very low proportion of rapid progressively (type A) motile spermatozoa of 5% in the fresh semen sample. Different conditions may impair sperm motility, but the aetiology of asthenozoospermia often remains unknown. Anti-sperm antibodies, infection of the genital tract, prolonged periods of anejaculation, high semen viscosity, ultrastructural defects and metabolic defects are the commonest causes (Vandervorst et al., 1997
). Some patients in this study had a high semen viscosity (5/20), and/or antisperm antibodies (2/20), and/or slightly elevated leukocyte counts (2/20). Although these parameters may impair motility or interfere with fertilization results after conventional IVF, the patients showing these defects were equally distributed over the fert and the fert+ groups (Table V
).
The most striking observation in the comparison between IVF and ICSI in this study population is the extremely low fertilization rate and high fertilization failure rate after conventional IVF. Once 2PN fertilization with conventional IVF had taken place, however, the rate was reasonably high (45.7%) but showed the tendency to be lower than the 2PN rate after ICSI (59.4%) for the same 10 patients. The observation that the 2PN rate after ICSI was 67.5% and 59.4% for the fert and the fert+ groups respectively, indicates that penetration through the oocyte investments after conventional IVF, rather than post-penetration events, are impaired in this study population. Differences in developmental speed (Table III) of the IVF and ICSI embryos are inherent to the ICSI technique itself. Nagy et al. (1998) showed that pronucleus formation after ICSI occurs 4 h sooner than after IVF due to the bypassing of several mechanical barriers. That is the reason why more IVF embryos than ICSI embryos were still at the 2-cell stage on the morning of day 2. In the present study, more ICSI embryos were replaced (36/43), even in the population that also achieved fertilization after IVF (fert+), which is mainly due to the later cleavage stage of the ICSI embryos at the time of embryo transfer, rather than to a difference in morphological quality. A high pregnancy rate was obtained. Four pregnancies, obtained in the patient population without fertilization after IVF, would not have occurred if ICSI had not been used. At least for these patients, the cycle was rescued by the application of combined ICSI and conventional IVF.
As half of the patients obtained fertilization only with ICSI and the other half with both ICSI and IVF, the two subpopulations were carefully explored. Semen samples with high viscosity, high antisperm antibody titres or leukocytes seemed equally distributed over the two subpopulations. Although each of these defects may interfere with fertilization after IVF, five out of 10 patients with fertilization after IVF had one of the three former defects, as well as five out of 10 patients with no fertilization after IVF. In terms of the basic sperm parameters, significant differences were observed for total sperm count and progressive motile sperm count on the day of oocyte retrieval. The progressively motile count ranged from 4.2x106 to 12.4x106 at oocyte retrieval for the fert group, and from 5.0x106 to 83.0x106 in the fert+ group. More relevant seems the quality of the sperm fraction prepared for insemination and injection, which was better in terms of motility for patients who obtained fertilization with both treatment procedures (fert+). Olds-Clarke (1996) demonstrated that penetration of the cumulus oophorus and especially of the zona pellucida require a certain velocity (VCL) of the spermatozoa in order to generate high forces. However, penetration of the egg investments seems to be not only dependent on sperm velocity but also on the quality of the investments, which may vary from one woman to another. This aspect may partly explain why here fertilization occurs after IVF in certain patients and not in others.
Furthermore, patients with fertilization failure after conventional IVF (fert) in the present study showed a high proportion of immotile spermatozoa (48%) in the inseminated sperm fraction, which was significantly different (P < 0.01) from that of the fert+ subpopulation (22%). In general, more than 80% progressive motility may be obtained after density gradient centrifugation. It is described in the literature, however, that high numbers of immotile and/or dead spermatozoa in the immediate vicinity of the oocyte may cause oxidative damage by the production of reactive oxygen species (Aitken and Clarkson, 1987; Aitken, 1994
). Oxidative stress may lead to impaired fertilization and/or embryonic quality, especially after prolonged sperm-to-oocyte exposure (Aitken and Clarkson, 1987
; Parinaud et al., 1993
; Aitken, 1994
; Gianaroli et al., 1998).
For several male patients in this study, asthenozoospermia (<5% type A motility) was not the sole sperm abnormality: it was often combined with teratozoospermia (<14% normal morphology). This additional defect may not be ignored as the fertilization rate after IVF is reported to be severely impaired in patients with less than 5% normal morphology (Kruger et al., 1988; Ombelet et al., 1994
; Payne et al., 1994
; Fishel et al., 1996). In the present study population, however, more semen samples from the fert+ group (6/10) showed severely defective sperm morphology (<5% normal forms) than did samples from the fert group (3/10). The question, however, may be raised as to whether `high insemination concentration', which may rescue the IVF cycle in severe cases of male infertility according to several studies (Oehninger et al., 1988
; Hammitt, 1993
; Ord et al., 1993
; Cowan et al., 1996
), might be helpful in avoiding fertilization failures for patients with extremely low or absent type A motility.
From the present study, it may be concluded that a low rate of rapid progressive sperm motility (<5% type A) is associated with a high rate of complete fertilization failures after conventional IVF, while the fertilization rates after ICSI are within the expected range. Although most parameters were similar, the subpopulation without fertilization after IVF showed a lower total sperm count and lower progressive motile count in the fresh semen than the subpopulation with fertilization after IVF. Of paramount importance seems to be the quality of the inseminating sperm fraction, with a higher rate of immotile spermatozoa and predominantly slow progressive motility in patients without fertilization after IVF. As the differences between the two subgroups were not clear-cut, it still remains to detect the underlying factors which might have a prognostic value for the success of conventional IVF. So far, however, these data argue for the application of a diagnostic combined conventional IVF/ICSI treatment cycle for male subfertility characterized by low or no rapid progressive motility, in order to avoid complete fertilization failure with conventional IVF.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 4, 1999; accepted on June 1, 1999.