Comparison between day-2 embryos obtained either from ICSI or resulting from short insemination IVF: influence of maternal age*

Yves Ménézo1,3 and Yona Barak2

1 Laboratoire Marcel Merieux, 1 Rue Laborde, 69500 BRON, France and 2 In Vitro Fertilization Unit, Herzliya Medical Center, 7 Ramot-Yam Street, Herzliya-on-Sea 46851, Israel


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Short incubation time prevents deleterious effects of cumulus cell degeneration and excess spermatozoa in IVF embryos. We performed a short incubation (3 h) protocol in 328 IVF cycles, in order to compare the developmental potential of regular IVF embryos with those originating from 316 cycles entered our intracytoplasmic sperm injection (ICSI) programme over the same period. Embryo transfers were performed in all patients on day 2. The mean number of embryos transferred was 1.92 for the ICSI group and 1.73 for the IVF group (P < 0.007). This was related only to the wishes of patients. However, the policy of the centre is to transfer a low number of embryos in young patients in order to avoid multiple pregnancies. All spare embryos were permitted to grow to the blastocyst stage for freezing. Shortening incubation time did not decrease fertilization rates. In our overall population, no difference was observed in the implantation rates per embryo for IVF (19%) or for ICSI (20%). An age-related decrease in embryo production was observed for both groups of patients (P < 0.01 for ICSI and P < 0.001 for IVF). The age-related decrease in embryo implantation was only significant for the IVF group (P < 0.03 for patients <30 and >35 years of age). A significant overall decrease in blastocyst formation was observed for spare embryos after ICSI versus IVF (34.2 versus 43.8%; P < 0.05). The significance of this observation is discussed.

Key words: embryo implantation/ICSI/IVF/short insemination time


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The paternal influence in embryogenesis has gained substantial attention since the appearance of techniques such as intracytoplasmic sperm injection (ICSI) (Palermo et al., 1992Go). Treatment of men with poor quality spermatozoa by using ICSI has improved our knowledge of sperm biology and several points of negative impact, both genetic and epigenetic, have been identified (Ménézo and Dale, 1994Go; Ménézo and Janny, 1997Go). A recent study (Shoukir et al., 1998Go) noted that ICSI embryos have a lower developmental potential as measured by blastocyst formation. Jones et al. (1998) also confirmed the negative impact of male factors on blastocyst formation. Gardner et al. (1998) were the only ones who could not detect this observation. One study (Gardner et al., 1998Go), however, did not identify a negative effect of ICSI on blastocyst formation.

In order to understand properly the effect of the ICSI technique on the developmental potential of ICSI compared with IVF embryos, we performed the short insemination time protocol (Gianaroli et al., 1996Go; Quinn et al., 1998Go) in regular IVF. In fact, in terms of culture conditions, the short insemination protocol is similar to that of ICSI; the oocytes are rapidly denuded, as after 3 h most of the cumulus cells are detached and removed by rinsing. They are no longer submitted to the possible deleterious effects of degenerating cumulus cells, or spermatozoa. We believe, therefore, that a comparison such as this will lead to a better understanding of the paternal effect on the developmental potential of the embryo. In addition, in order to discriminate a possible maternal effect, the patients were ranked by partner ages: <30, 30–35 and >35 years, as the age of 30 was shown as a `shifting point' in fertility (Piette et al., 1990Go; Janny and Ménézo, 1996Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study, undertaken from April 1998 to the end of December 1998, was performed on patients who entered the IVF programme at the Institut Rhonalpin, Laboratoire Marcel Merieux in Bron (France). A total of 644 cycles was assessed, of which 316 were ICSI. In 328 IVF cycles a short insemination procedure took place during conventional IVF. IVF was performed on the first attempt when >1.5x106 spermatozoa with at least 50% motility could be observed in the specimen. ICSI was performed: (i) when the concentration of motile spermatozoa was <1.5x106 in the total ejaculate; (ii) when the percentage of abnormal forms was over 90% according to the World Health Organization criteria (WHO, 1992); (iii) after one previous fertilization failure with no spermatozoa attached to the zona pellucida in one IVF cycle, in which at least three metaphase II (MII) oocytes had been retrieved.

This proportion of ICSI/IVF corresponds to our regular activity. The patient population in the ICSI group for ages <30, 30–35 and >35 years comprised 94, 140 and 82 cycles respectively totalling 316, and in the IVF group there were 61, 143, and 124 cycles respectively, totalling 328 in all.

The stimulation protocol was identical to that already described (Janny and Ménézo, 1994Go). Briefly, it included the use of gonadotrophin-releasing hormone analogues (GnRHa) in semi-long and/or short protocols, followed by stimulation with urinary follicle stimulating hormone (uFSH), or recombinant FSH (rFSH). Ovulation was triggered between day 11 and day 13 of stimulation, by 9000 IU of human chorionic gonadotrophin (HCG). The oestradiol concentration at the time of ovulation was ~150 pg/ml/follicle.

IVF procedure and culture media
The follicle-rinsing medium was prepared in the laboratory, i.e. Earle's medium (Sigma, St Quentin Fallavier, France) supplemented with 0.4% of human serum albumin (LFB, 78000, Les ULIS, France) and 80 mg/l of gentamycin. The same medium was used for washing the spermatozoa which were processed by a two-layer pure-sperm gradient (IVF Science, Gothenburg, Sweden) for ICSI and regular IVF.

Insemination of oocytes took place in 0.5 ml Universal-IVF (U-IVF, Medicult, Copenhagen, Denmark), in a 4-well dish (Nalge Nunc, Roskilde, Denmark). Three hours later, inseminated oocytes were placed in a fresh droplet of U-IVF medium. At this point, the spermatozoa and >90% of the cumulus were naturally removed. Only a few corona cells still surrounded the oocytes. Eighteen hours later, the oocytes were examined for the presence of pronuclei. As the oocytes were usually devoid of the majority of cumulus cells, a slight motion in a pipette totally removed the remaining corona cells around the zygotes. Observation of the fertilized oocytes was therefore easy and fast.

Zygotes were then transferred into fresh droplets of Universal IVF medium (Medi-Cult). Embryo transfer took place 44–48 h post-insemination. Supernumerary embryos were grown in a sequence of media similar to those previously described (Chouteau et al., 1998Go). Embryos that reached the blastocyst stage were frozen according to a protocol previously described (Ménézo et al., 1996Go; Ménézo and Veiga, 1997Go). All procedures were performed in droplets under oil (light oil; BDH, Poole, Dorset, UK), in a 5% CO2 atmosphere in air.

ICSI
Within 1 h after ovum retrieval, oocytes were denuded in a solution of 80 IU hyaluronidase/ml BM1 medium (Ellios Biomedia, Paris, France). Sperm microinjection was performed in droplets under oil of BM1 medium containing 10% PVP, and the injected oocytes were then cultured in U-IVF (Medicult), for the subsequent 48 h. Fertilization was checked 20–22 h later in the same droplets. The remaining supernumerary embryos were cultured for freezing at the blastocyst stage. As mentioned above, all the procedures were performed under oil, in a 5% CO2 atmosphere in air.

Embryo transfer
All embryo transfers were performed in BM1 medium. Oocyte collection, maturation, fertilization, pregnancy rates per transfer and the implantation rates per embryo were recorded.

Statistical analysis
As it was not possible to determine precisely the number of MII oocytes in IVF, where they were cumulus-enclosed, in contrast to ICSI where MII oocytes had had cumulus cells removed, all calculations were made considering the number of collected cumulus–oocyte complexes (COC).

Statistical analysis was carried out using SPSS software. General descriptive statistics were performed for the entire population and Student's t-test was used to compare the two treatments.

Quantitative data are presented as mean ± SD. Overall comparisons of means between the various groups were processed by analysis of variance (ANOVA), and two-by-two comparison by t-test was used to determine which groups were significantly different.

For qualitative data, the overall comparisons of the distribution between the groups were processed by {chi}2 analysis. When a difference was found, a two-by-two comparison by {chi}2 was used to determine which groups were significantly different.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The entire population analysis was performed using ANOVA. Pairwise comparisons using the t-test were made in cases of significant F. The t-test that compared the whole popula- tion showed a higher maternal age for the IVF group: 33.93 ± 4.69 versus 32.43 ± 4.45 years for IVF and ICSI groups respectively (P < 0001).

The mean ages for the three IVF versus ICSI groups were as follows: (i) <30 years, 27.35 ± 1.86 versus 27.69 ± 1.53; (ii) 30–35 years, 32.45 ± 2.78 versus 32.17 ± 1.59; (iii) >35 years, 38.24 ± 3.87 versus 39.03 ± 3.31.

As shown in Figure 1Go, in the ICSI-treated group, the number of COC significantly decreased in the oldest group (>35 years), in comparison with the two other age groups (P < 0.005). In the IVF group the decrease was significant between all three age groups (P < 0.0001). In both ICSI and IVF groups, an age-related decrease was also found in the number of embryos obtained. A higher embryo formation, per collected COC, was observed for IVF (P < 0.0005; Figure 2Go). The mean number of embryos obtained was higher for the IVF group (P < 0.0001; Table IGo).



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Figure 1. Mean ± SD number of cumulus–oocyte complexes (COC) collected from ICSI and IVF patients in the various age groups. In the ICSI-treated group, the number of COC significantly decreased in the oldest group (>35 years), in comparison with the two others (P < 0.005). In the IVF group the decrease was significant between all three age groups; P < 0.0001). Values shown above columns are mean number of COC.

 


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Figure 2. Ratio of number of embryos formed and number of cumulus–oocyte complexes (COC) in the ICSI and IVF populations of the various age groups. A higher ratio was noticed in the IVF group overall (P < 0.0005). No difference was observed in the ratio when various age groups were compared within the entire population. However, a decrease in embryo formation was noticed in the older ICSI groups (age 30–35 and >35 years), in comparison with the younger group (age <30 years); P < 0.01; this was not observed in the IVF group. Values above columns are ratios.

 

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Table I. A t-test comparison between ICSI versus IVF in the entire population
 
The mean number of embryos transferred was lower in the IVF group (1.7 versus 1.9; P < 0.007), but the pregnancy rate per transfer and the implantation rates per embryos were similar (Table IGo). No difference was noticed in the mean number of embryos transferred, by age group.

Differences were found between pregnancy rates (Figure 3Go) in the young group (42.6%), the 30–35 year group (31.2%) and the group of >35 years (24.8%; {chi}2 = 12.95; P < 0.001), in the entire population. Differences were also found in implantation rates between the youngest group and that of the >35 group (25 versus 16% respectively; P < 0.02; Figure 4Go). No significant differences were found in pregnancy or implantation rates when the ICSI and IVF groups were compared (Figures 3 and 4GoGo).



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Figure 3. Pregnancy rates: a comparison of pregnancies per embryo transfer among the various age groups in the ICSI and IVF cycles. No difference was observed between the ICSI versus IVF cycles. Comparisons between the various age groups in the entire population showed differences between the groups ({chi}2 = 12.95; P < 0.001), with the pregnancy rate of the youngest group being significantly higher than the others. A comparison among the various age groups within the ICSI population showed differences between the youngest group and the oldest (>35 years) group; {chi}2 = 6.6; P < 0.01). Values above columns are pregnancy rates.

 


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Figure 4. Implantation rate: number of implanted embryos per number transferred in the various age groups of the ICSI and IVF populations. Implantation rates were similar when the ICSI and IVF groups were compared. A comparison between the various age groups in the entire population showed a higher implantation rate for the <30 years group (P < 0.02). No difference was noticed between the various age groups within the ICSI population. A higher implantation rate was observed in the <30 years age group in the IVF population (P < 0.03). Values shown above columns are implantation rates.

 
No differences in implantation rate were noticed between the various age groups within the ICSI population (Figure 4Go). However, a higher implantation rate was observed for the youngest group (<30 years) compared to the oldest group (>35 years) in the IVF patients (P < 0.03).

Regression and discriminate analyses gave no indication or model to predict success. Although it was not statistically significant, we observed an age-related increase in polyploidy for IVF embryos, that reached 10.7% for patients >35 years of age.

A significant decrease in blastocyst formation was observed in the supernumerary ICSI versus IVF embryos [130/380 (34.2%) and 543/1241 (43.8%) respectively; P < 0.05].


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The short insemination method did not decrease fertilization and cleavage rates. In 1997, during a corresponding period of time, when oocytes of 538 cycles were incubated with spermatozoa overnight as a routine procedure for insemination, our overall cleavage rate was 61%. In our current study, the cleavage rate was 57% in the 328 cycles, which were treated by the short insemination protocol. This confirms the results from previous short co-incubation time studies of oocytes and spermatozoa for fertilization (Gianaroli et al., 1996Go; Quinn et al., 1998Go). In general, for IVF embryos the polyploidy observed by us, of up to 10.7% for patients >35 years of age, is rather higher than that currently described in the literature (8–10%) in this age range (Ho et al., 1994Go). Spermatozoa remained in contact with the oocytes for a shorter period, and most cumulus cells were removed while rinsing the oocytes 3 h post-insemination. Thus, the zygotes are `cleaner' and a simple motion with the denuding pipette retrieves the few remaining corona cells. This smooth removal may also have a positive impact on embryo quality.

The age-related decline in ovarian production, determined by the number of COC collected, has been previously observed (Piette et al., 1990Go; Janny and Ménézo, 1996Go; Figure 1Go). A higher implantation rate was observed as an outcome of IVF with short co-incubation time, when compared with the results of routine IVF (e.g. overnight incubation with spermatozoa) in the previous year during the same period of time (19% versus 13.5%) in our Unit, (Dossier FIVNAT 1999Go).

The possibility that reduced zona hardening may facilitate hatching and implantation has previously been mentioned (Waldenstrom et al., 1993Go). It is possible that the early removal of excess spermatozoa may avoid exposure of the embryos to a high density of free radicals generated by supernumerary spermatozoa and degeneration of cumulus cells. Reactive oxygen species induce zona hardening and tanning. Apart from this aspect, they also have a deleterious effect on DNA. With regard to these biochemical aspects, the short insemination time is the only protocol which makes it possible to compare ICSI and IVF embryos. Our data show no difference in pregnancy outcome between IVF and ICSI embryos in the overall population.

The implantation rates per embryo of ~19% were similar for ICSI and IVF embryos in our overall population. At first sight, this similarity is surprising: epigenetic-related problems, such as centrosome function or dysfunction, (Sathananthan et al., 1996Go; Van Blerkom, 1996Go) are expected to interfere with embryo quality. As mentioned earlier (Ménézo and Janny, 1997Go), ICSI may rescue embryos in some patients by avoiding delays in cell cycles through a direct sperm deposition in the oocyte. The spare time helps to save maternal messenger ribonucleic acid (mRNA), which may partially overcome any epigenetic defects. This agrees with previous observations (Oehninger et al., 1996Go) that ICSI embryos, in cases of severe teratozoospermia, have higher morphological scores and higher developmental potential than their corresponding IVF embryos. Moreover, as fast-cleaving embryos lead to the best pregnancy rates after transfer on day 2 or 3, as observed (Shoukir et al. 1997Go), this aspect was confirmed.

Our lower embryo cleavage rate was observed for ICSI in this study, which is either related to technical problems, or to these epigenetic or genetic problems. It has also been observed (Asch et al., 1995Go) that a considerable proportion of the so-called unfertilized eggs are fertilized, but are unable to perform even the first division. In ICSI, the quality of the injected spermatozoon is randomly chosen; selection is performed on gross morphological aspect and motility (when possible). These sperm characteristics probably have nothing to do with the real potential of the spermatozoon, in terms of affecting the developing embryo. It is likely, therefore, that only some of the injected spermatozoa have a good developmental potential (Hewitson et al., 1997Go). Moreover, Lundin et al. (1998) claimed that ICSI can increase genetic risk (Lundin et al., 1998Go). Although ICSI is performed to increase the chance of fertilization, even with good spermatozoa, in most cases the real genetic capacity of the spermatozoon is unknown. It is admitted (Lundin et al., 1998Go) that, when possible, it is better to perform regular IVF in order to try and avoid genetic risk, as again nothing is known about the injected spermatozoon, suggesting that a study such as the current one to compare ICSI and IVF would be useful. Obviously it would be necessary, ideally, to compare ICSI and IVF in the case of good spermatozoa. This would facilitate determination of the contribution related to the extra manipulation of the embryos due to the injection only, in cases of ICSI, and the putative damage to the spindle, that follow (Hewitson et al., 1999Go). However, this kind of investigation is ethically questionable.

Thus, we assume that, in comparison to IVF, fewer embryos may reach the blastocyst stage in ICSI. In addition, when embryo transfers are performed on day 2, as in the study presented here, a partial selection of embryos based on cleavage speed and morphological appearance also occurs on that day. Therefore, even fewer ICSI embryos will be able to reach the blastocyst stage when compared to regular IVF.

Our data on spare embryos to some extent agree with these predictions. This simply indicates that not all the epigenetic and genetic problems mentioned earlier, and those related to poor sperm quality, can be overcome. The rescue is only partial for the overall ICSI embryo population. This is also in agreement with the observations of Shoukir et al. (1998) concerning decreased blastocyst formation for ICSI embryos, and it confirms the paternal effect on early embryogenesis (Janny and Ménézo, 1994Go; Jones et al., 1998Go). Moreover, the apparently (but not significantly) lower implantation rate per embryo observed for ICSI versus regular IVF in younger patients (Figure 4Go) (aged <30 years) may also be linked to a paternal effect in these couples, where the maternal effect is minimal.

According to the above data, we confirm that obtaining early-stage embryos does not necessarily lead to a positive end-point, and furthermore, ICSI cannot universally cure major sperm defects.

A study of blastocyst formation is currently taking place for ICSI and IVF patients who enter our blastocyst transfer programme. This will facilitate ultimate determination of whether blastocyst formation is impaired in ICSI patients, and whether blastocysts obtained for IVF and ICSI have the same developmental (implantation) potential.


    Acknowledgments
 
Our thanks to Mrs Sara Intrater for statistical assistance.


    Notes
 
3 To whom correspondence should be addressed at: Laboratoire Marcel Merieux, 1 Rue Laborde, 69500 Bron France, France.E-mail: yves.menezo{at}insa-lyon.fr Back

* Presented in part at the 1999 American Society for Reproductive Medicine, September 25–30, 1999, Toronto. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on September 30, 1999; accepted on April 26, 2000.