Conventional in-vitro fertilization versus intracytoplasmic sperm injection in sibling oocytes from couples with tubal infertility and normozoospermic semen

Catherine Staessen1, Michel Camus, Koen Clasen, Anick De Vos and André Van Steirteghem

Centre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, Brussels, Belgium


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An auto-controlled study was conducted in couples with tubal infertility and normozoospermic semen. The fertilization rates and embryonic development in sibling oocytes treated, using the same semen sample, either by conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) at the same time were compared. Sibling oocyte–cumulus complexes (OCC) of 56 different couples with tubal infertility and normozoospermic semen were randomly divided in order of retrieval into two groups inseminated either by conventional IVF or by ICSI. Of the retrieved OCC in the same cohort, 53.0 ± 31.2 and 62.0 ± 26.6% showed two distinct pronuclei after conventional IVF and ICSI respectively (not significant). Complete fertilization failure occurred after conventional IVF in 12.5% (7/56 couples). After ICSI, the comparable figure was 3.6% (2/56). The number of cases was too small to apply a statistical test to this difference. Total cleavage rates were quite similar: 86.7 ± 28.0 and 90.1 ± 21% of the zygotes developed into transferable embryos after IVF and ICSI respectively (not significant). Similarly, no difference in embryo quality was observed. Although injection and insemination of the oocytes were performed at the same time in the two groups, at 42 h post-insemination more embryos were at the four-cell stage after ICSI (P < 0.001) than after conventional IVF, where more embryos were still at the two-cell stage (P < 0.02). Embryo transfer was possible in all 56 couples, resulting in 16 positive serum human chorionic gonadotrophin tests (28.6% per embryo transfer), from which a clinical pregnancy resulted in 15 couples. The best embryos were selected for transfer independently of the insemination procedure, but preferably from the same origin. There appeared to be no difference in implantation potency of the embryos obtained with either technique after the non-randomized transfers.

Key words: IVF versus ICSI/sibling oocytes/tubal infertility


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Intracytoplasmic sperm injection (ICSI) was introduced initially into clinical practice to treat couples with severe male factor infertility, who could not be helped by conventional in-vitro fertilization (IVF) because they had too few progressively motile spermatozoa (Palermo et al., 1992Go; Van Steirteghem et al., 1993Go, 1998Go). The development of ICSI also opened new treatment perspectives for patients with obstructive or non-obstructive azoospermia (Nagy et al., 1995aGo). Acquired scientific knowledge, increasing technical experience and further adaptation of the sophisticated microtools resulted in improved fertilization and embryo development rates. The rates of embryo development and pregnancy obtained with the ICSI technique now reach those obtained with conventional IVF (Staessen et al., 1995Go); although used for different types of infertility. The question arises as to whether ICSI might be beneficial even in cases where no male factor is involved.

An auto-controlled study was conducted in couples with tubal infertility and normozoospermic semen, in order to compare fertilization rates and embryonic development in sibling oocytes treated simultaneously either by conventional IVF or by ICSI using the same semen sample.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovarian stimulation
All patients received ovarian stimulation treatment in order to provide multiple oocytes. Ovarian stimulation was performed by a desensitizing protocol using gonadotrophin-releasing hormone agonist (GnRHa) in association with human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) (Ubaldi et al., 1995Go). Oocyte retrieval was carried out by ultrasound-guided transvaginal puncture 36 h after HCG administration.

Study design
From September 1996 until August 1998, couples were selected at the fertility clinic according to the following criteria: (i) tubal infertility, (ii) normozoospermic semen with a sperm count of >=10x106 sperm cells/ml, >=8% morphologically normal sperm cells (Kruger's strict criteria) and >=40% progressively motile sperm cells. Informed consent was obtained from all couples involved in the study. If at the moment of oocyte retrieval at least six oocyte–cumulus complexes (OCC) were obtained, the patients effectively entered the study.

Patient characteristics
In 56 couples entering the study, the mean age of the women was 33.1 ± 4.4 years and of the men 34.3 ± 5.2 years. It was a first trial in 29 couples, a second trial in 15 couples and a third in 12 couples. All 27 couples with a previous trial had achieved fertilization following IVF.

Sperm preparation
Spermatozoa were prepared either by swim-up or by centrifugation using a discontinuous gradient (initially Percoll, later Puresperm). After preparation, the same semen sample was used for conventional IVF and for ICSI.

Oocyte retrieval, insemination or injection
At the moment of oocyte retrieval, the sibling OCC of the 56 different couples were randomly divided in order of retrieval into two groups. From both groups, the OCC were placed in 25 µl droplets of Ménézo B2 medium (bioMérieux, Montalieu Vercieu, France) covered by lightweight paraffin oil and incubated in a humidified 37°C incubator (5% CO2, 5% O2 and 90% N2). All OCC in one group were treated by conventional IVF and were inseminated as described previously (Staessen et al., 1995Go). The other group of OCC was treated by ICSI: after removal of the cumulus cells the metaphase II oocytes were injected as described previously (Van Steirteghem et al., 1995Go). Both procedures were performed simultaneously, ~4 h after oocyte retrieval.

Pronuclei and embryo evaluations
Pronuclei evaluation was carried out 12–18 h later. Embryonic development was assessed 24 h later (42 h after insemination or injection). The embryos were classified according to a simplified system for morphological criteria: (i) type A embryos in which all blastomeres were of an approximately equal size and without anucleate fragments, (ii) type B embryos had blastomeres of equal or unequal size and had <20% of the volume of the embryo filled with anucleate fragments and (iii) type C embryos with anucleate fragments occupying between 20 and 50% of the volume of the embryo. At this juncture, independently of the insemination procedure, the best embryos in terms of morphological appearance and rate of development were selected for transfer.

Embryo transfer
The transfer was performed about ±48 h after insemination or injection. To reduce the risk of high-rank multiple pregnancies, the number of embryos replaced was mostly limited to two or three.

Cryopreservation
Spare embryos of sufficient morphological quality were cryopreserved (Van den Abbeel et al., 1997Go).

Pregnancy
Pregnancy was confirmed by detecting rising HCG concentrations on at least two occasions at least 12 days after embryo replacement. Clinical pregnancy was determined by the presence of gestational sac by echographic screening at 7 weeks or later. The implantation rate was calculated as follows: preclinical abortions were reconsidered as a minimum of one implantation; clinical abortions, EUG and ongoing pregnancies were reported as implantation by the number of fetal sacs observed. As ongoing implantation, the number of implantations developing beyond 12 weeks was counted.

Statistical analysis
The two-tailed paired t-test and contingency table were applied for statistical analysis. Significance was defined as P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In these 56 cycles, a total of 662 OCC were collected (mean of 11.8 ± 4.4 OCC per cycle); 334 sibling OCC were inseminated by conventional IVF (a mean of 6.0 ± 2.2 OCC per couple) and the remaining 328 (a mean of 5.9 ± 2.3 OCC per couple) were treated by ICSI (Table IGo). The sperm characteristics of the ejaculated sample used for the oocyte insemination and injection were as follows: (i) a mean count of 69.2 ± 48.9x106 sperm cells/ml and (ii) a mean of 48.9 ± 10.1% motile sperm cells. The morphology was not rechecked on these sperm samples but was normal at the time of patient enrolment.


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Table I. 2PN-fertilization in sibling oocytes treated by conventional IVF or by ICSI
 
In the same cohort of OCC, 53.0 ± 31.2% of the retrieved OCC showed two distinct pronuclei after the treatment by conventional IVF and 62.0 ± 26.6% of the retrieved OCC after ICSI (not significant; two-tailed paired t-test). From the oocytes treated by ICSI, the cumulus cells were removed prior to the microinjection procedure. In 266 out of the 328 (81.1%) OCC retrieved, a metaphase II oocyte was observed and injected; giving a normal fertilization rate of 77.7 ± 28.0% per injected oocyte. For the comparison between IVF and ICSI, fertilization is expressed as the number of normally fertilized oocytes per number of retrieved OCC.

Complete fertilization failure occurred after conventional IVF in seven couples (41 OCC; a mean of 5.9 ± 1.8 inseminated OCC) out of the 56 (12.5%); in six couples this occurred in a first treatment cycle and in one patient in a second trial. After ICSI, on the other hand, complete fertilization failure occurred in only two couples (a mean of 3.5 ± 0.7 OCC; a total of seven OCC containing only three MII oocytes) out of the 56 (3.6%). Considering the 47 couples achieving fertilization with both techniques, a similar fertilization rate was obtained: 60.3 ± 25.6% of the IVF-inseminated oocytes showed two pronuclei versus 63.8 ± 25.2% of the ICSI-treated oocytes.

The total cleavage rates were quite similar; after IVF 87% of the zygotes developed into transferable embryos (type A, B and C) and after ICSI 89.5% (two-tailed paired t-test; not significant). Similarly, no differences between the proportions of type A, B and C embryos were observed (Table IIGo).


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Table II. Comparison of embryonic cleavage and development in 47 couples with fertilization after ICSI and after conventional IVF
 
Although injection and insemination of the oocytes were performed simultaneously, 42 h later more embryos were at the four-cell stage after ICSI (two-tailed paired t-test; P < 0.001) and more embryos were still at the two-cell stage after conventional IVF (two-tailed paired t-test; P < 0.02) (Table IIGo). The high standard deviation indicated that between the different cycles, a high variation in the stage of embryo development at 42 h after insemination was observed. Within the cycles, however, a consistent difference in developmental stage between IVF and ICSI was observed.

In our IVF–ICSI programme, the best embryos in terms of morphological appearance and rate of development were selected for transfer on the basis of the first embryo evaluation, explaining the higher number of ICSI compared with IVF embryo transfers (Table IIIGo). The best embryos were selected for transfer independently of the insemination procedure, but preferably involving only one type of insemination. A transfer was performed in all 56 couples, resulting in 16 positive serum HCG tests (28.6% per embryo transfer) from which 15 clinical pregnancies resulted. Randomly, 13 transfers only involved conventional IVF, 27 transfers involved only ICSI embryos and 16 transfers involved a mixture of conventional IVF and ICSI embryos. The outcome of the transfers is presented in Table IIIGo. The numbers of positive HCG measurements and ensuing clinical pregnancies were quite similar across the three groups (not significant; contingency table). The implantation rate per embryo and the ongoing implantation rate (12 weeks of pregnancy was the endpoint) are not different. Furthermore, of 114 embryos cryopreserved, 62 were obtained after IVF and 52 after ICSI.


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Table III. Outcome of the fresh embryo transfers (n = 56)
 
To evaluate the data for conventional IVF obtained in the present study, we compared with the results from 112 consecutive different couples with tubal infertility and normozoospermic semen treated by conventional IVF during the study period in our centre (Table IVGo). If the same couple underwent several cycles during the study period, only the first treatment cycle was considered. The couples' characteristics were similar to those of the patients involved in the study. In 65 couples this was a first treatment cycle and 47 had had previous cycles with fertilization after conventional IVF. For these couples, a fertilization rate of 59.4 ± 29.2% was obtained. Complete fertilization failure was observed in nine out of the 112 couples (8%), five couples during their first treatment cycle and four couples in their following cycle. A positive serum HCG was observed in 37 out of the 102 embryo transfers (36.3% per transfer). At the time of writing, the outcome of all the pregnancies is not yet known, and so 12 weeks of gestation was taken as the endpoint. From the 37 couples with a positive HCG, seven ended in a preclinical abortion, six in a clinical abortion and one in an ectopic gestation; 23 pregnancies developed beyond 12 weeks of gestation. Of the transferred embryos, 17.5% (47/269) implanted and 10.8% (29/269) gave rise to a fetus developing beyond 12 weeks.


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Table IV. Clinical results of 112 different couples with tubal infertility and normozoospermic semen treated by conventional IVF during the study period
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of our study indicate that in couples where no male factor infertility was expected, the fertilization rate in sibling oocytes inseminated by conventional IVF or by ICSI was not significantly different. We found ~9% difference in favour of ICSI, but there was a high non-consistent variation between the patients. After conventional IVF, a complete fertilization failure occurred in 12.5% of the couples, while this occurred in only 3.6% of the couples when oocytes were microinjected.

Similar studies comparing IVF versus ICSI in sibling oocytes from couples with unexplained fertility and non-male factor have been previously performed (Aboulgar et al., 1996a; Yang et al., 1996Go; Ruiz et al., 1997Go). In the study by Yang et al. (Yang et al., 1996Go), 13 couples were included yielding a fertilization rate of 61% after conventional IVF and 67% after ICSI (not significant). No significant difference in fertilization rates between ICSI (50.0%) and IVF (50.7%) was found in 22 couples (Aboulgar et al., 1996). Similarly, another study of 70 patients (Ruiz et al., 1997Go) found no significant difference in the fertilization rates between IVF (54.0%) and ICSI (60.4%). Our results relate well with these studies, showing also an apparently higher fertilization rate after ICSI but never reaching the level of statistical difference.

Unlike the study by Yang et al. (Yang et al., 1996Go), the other two studies include respectively 22.7 (Aboulgar et al., 1996a) and 11.4% (Ruiz et al., 1997Go) of the couples with unexplained infertility with completely unexpected fertilization failures after conventional IVF. In none of these three studies were there complete fertilization failures after ICSI.

In patients with tubal infertility it is primarily a mechanical barrier to sperm–oocyte contact that causes their infertility. Since exposure of the oocytes to the spermatozoa was impossible, proof of the fecundity of the apparently normal spermatozoa is absent. During the IVF procedure, the mechanical barrier is removed and the fecundity of the spermatozoa can be proven in vitro. The results demonstrate that fertilization does not always follow. Unexpected fertilization failures after IVF in couples with tubal infertility and normozoospermic semen such as we are dealing with in the present study have been described previously reaching ~8% (Barlow et al., 1990Go; Lipitz et al., 1994Go). Undefined and repetitive sperm–oocyte interaction defects may also be present in such tubal patients. Comparing conventional IVF and ICSI on the sibling oocytes for diagnostic purposes in the first treatment cycle would detect such couples.

However, previous studies have shown that complete failure of fertilization in most couples with normal sperm parameters and infertility due to a tubal factor is associated with excellent prognosis for subsequent IVF cycles (Molloy et al., 1991Go; Lipitz et al., 1994Go) with an equally favourable outcome. Unexpected complete fertilization failure after IVF, is not only limited to first trials. In the present study of the seven couples with failed fertilization, six were in a first cycle and one in a third cycle (normal fertilization in previous cycles). Similarly, of the nine out of 112 cycles with failed fertilization a previous cycle with a good fertilization rate was documented in four of them. It is therefore frustrating that from one treatment cycle to another, the fertilization rate after conventional IVF can vary from 0 to 100%. This demonstrates the complexity and the sensitivity of the cascade of events leading to normal fertilization. Complete fertilization failure in these couples may be due to non-repetitive factors such as treatment-related inadequate ovarian stimulation producing fewer good oocytes or temporarily inadequate laboratory conditions.

The risk of complete fertilization failure was minimized after ICSI; none had been found in previously reported studies (Aboulgar et al., 1996a; Yang et al., 1996Go; Ruiz et al., 1997Go) and we found complete failure after ICSI, in 3.5% of the couples in cases where only a few oocytes were available for injection. Complete fertilization failure after ICSI may be associated with low numbers and poor quality of oocytes (Liu et al., 1995Go).

Reinsemination of the unfertilized oocytes by ICSI might be considered as a method by which to rescue failed conventional IVF cycles. Although ICSI rescue of unfertilized oocytes is possible in terms of fertilization (Nagy et al., 1995bGo), the normality of the genetic status of the embryos (Nagy et al., 1995bGo) is in question some liveborn children have, nevertheless been reported (Morton et al., 1997Go).

Another point of interest is the fact that in ICSI the oocytes are denuded before injection, allowing the nuclear maturity of the oocytes to be determined and the injection to be performed after first polar body extrusion. This allows evaluation of the stimulation regimen of the patients so as to adapt it for possible new trials. In conventional IVF, oocyte maturity is usually estimated only on the basis of the OCC.

In couples with fertilization after both IVF and ICSI, an approximately equal fertilization rate of ~60% per retrieved OCC was obtained with both techniques. This may suggest that of the retrieved oocytes, only 60% are in fact fertilizable. It has already been shown that after cumulus removal only 80% are indeed at MII. Investigation of unfertilized oocytes after ICSI has shown that a technical problem of deposition of the spermatozoon inside the egg is not the major cause, but rather a failure to complete both the maternal and paternal chromatin transitions associated with normal fertilization (Dubey et al., 1997Go). The same phenomenon might be expected after failure of conventional IVF with competent fertilizing spermatozoa.

No differences in embryo morphological quality were found, indicating that subsequent morphological development was similar for zygotes obtained after ICSI or after IVF. The micromanipulation seems to have no effect on the morphological development of the embryos. Most previous IVF-versus-ICSI comparative studies on sibling oocytes found no difference in embryo quality (Calderon et al., 1995Go; Ruiz et al., 1997Go) with the exception of one study (Yang et al., 1996Go) which found a higher percentage of type A embryos in the ICSI-treated oocytes on the basis of only 13 cases.

Since no difference in embryo quality was observed between IVF- and ICSI-generated embryos, no support can be provided for the hypothesis that long exposure of oocytes to large numbers of spermatozoa creating suboptimal culture conditions by the extensive generation of reactive oxygen species leads to inferior embryo quality (Quinn et al., 1998Go).

About 42 h after injection or insemination, a significantly higher proportion of embryos reached the four-cell stage in the ICSI group, indicating that they were more advanced. In ICSI, the spermatozoon is injected directly into the cytoplasm, reducing the time for pronucleus formation (Balakier et al., 1993Go; Nagy et al., 1998Go). These data indicate a consequent earlier start of embryonic development, since such embryos are in a more advanced stage than the embryos obtained after conventional IVF. Moreover, after conventional IVF, the exact timing of fertilization is not known, as opposed to ICSI, which results in an almost synchronized population of activated oocytes (Dozortsev et al., 1995Go). If we assume a synchronized start of embryo development, it is easier to distinguish more slowly developing embryos from normally developing embryos after ICSI. We selected the more advanced embryos for each transfer and preferably those produced by only one type of insemination technique, resulting in more transfers with embryos obtained after ICSI.

With regard to an effect on pregnancy and implantation rates, not enough data have been obtained to be conclusive. In one larger study, involving 116 patients with tubal infertility (Aboulghar, 1996bGo), 58 were treated randomly with IVF and 58 with ICSI and no difference in pregnancy rate was found.

An important question arises as to whether ICSI will become the procedure of choice for all couples requiring assisted reproduction treatment. Without doubt, the ICSI procedure itself is more invasive. Therefore questions with regard to safety issues have to be raised. In a recent publication (Munné et al., 1998Go), no statistical differences were observed between the rates of abnormalities among embryos generated by ICSI or conventional IVF. On the other hand, a follow-up study involving 1082 karyotypes of ICSI children demonstrated a slightly increased incidence of chromosomal abnormalities (Aytoz et al., 1998Go; Bonduelle et al., 1998Go). It remains to be seen whether these findings are linked to the technique itself or to the severe andrological infertility of the treated patients. So far, no large-scale well-documented data on the outcome of children after ICSI with normozoospermic semen are available.

In conclusion, fertilization rates after IVF and ICSI are comparable but ICSI offers the advantage of minimizing the risk of complete fertilization failure. This study provides no information to the detriment of ICSI in comparison with conventional IVF in regard to embryonic development and implantation potential. A possible strategy, however, might be to compare IVF versus ICSI in all first treatment cycles. This would certainly be helpful in cases where a structural repetitive fertilization problem is present. However, typical cycle-related fertilization failures will not be avoided in this way.


    Acknowledgments
 
The authors wish to thank the clinical, scientific, nursing and technical staff failure of the Centre for Reproductive Medicine. We thank Frank Winter of the Language Education Centre for correcting the English text. Grants from the Belgian Fund for Medical Research are gratefully acknowledged.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 26, 1998; accepted on June 21, 1999.