1 Department of Obstetrics and Gynaecology and 2 Department of Molecular Cell Biology and Genetics, Academic Hospital, University of Maastricht, Maastricht, The Netherlands
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Abstract |
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Key words: human blastocysts/ICSI/preimplantation development
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Introduction |
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In spite of these reassuring results, concern has been raised about the potential dangers of the ICSI procedure. Theoretically, both the injection technique itself as well as the possible injection of abnormal spermatozoa can affect oocyte and subsequent embryo quality. During injection, it cannot be avoided that a small quantity of medium, which contains potentially harmful components, is injected into the oocyte. Also, physical damage to cytoplasmic structures can possibly be inflicted by a traumatic injection.
In view of these possible risks, it may be postulated that zygotes originating from ICSI have a higher incidence of sublethal cell damage and subsequent impaired embryonic development as compared to zygotes originating from conventional IVF. In this study, we present a retrospective analysis of the development in vitro of embryos derived from conventional IVF in comparison with ICSI. Developmental stages and morphological qualities of surplus embryos at the second and third day after insemination or injection, and embryonic development to the blastocyst stage were assessed.
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Materials and methods |
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The stimulation protocol used has been described previously (Land et al., 1996). In summary, the gonadotrophin-releasing hormone (GnRH) agonist nafarelin (Synarel; Searle BV, Maarssen, The Netherlands) was used in combination with human menopausal gonadotrophin (HMG, Pergonal; Serono, Amsterdam, The Netherlands; or Humegon; Organon, Oss, The Netherlands) to stimulate multiple follicular development. Follicle growth was monitored by ultrasound and 5000 IU of human chorionic gonadotrophin (HCG, Pregnyl; Organon) was given as soon as the dominant follicle was judged to be mature (>18 mm diameter), to induce final follicular and oocyte maturation. Ultrasound-guided oocyte retrieval was performed 3436 h after HCG administration. Insemination or ICSI was performed ~5 h after oocyte retrieval.
IVF procedure
Oocytes were inseminated with a calculated volume of a swim-up sperm suspension which resulted in a concentration of ~50 000/ml progressively moving spermatozoa as described (Enginsu et al., 1992). After incubation for 1820 h, corona cells that remained attached to the oocyte were removed using drawn Pasteur pipettes. Oocytes were checked for the presence of pronuclei, washed once, and transferred to fresh medium.
ICSI procedure
For the ICSI procedure, a previously published protocol was followed (Van Steirteghem et al., 1993). Briefly, after oocyte retrieval, cells of the cumulus and corona were removed by incubation for 3060 s in HEPES-buffered human tubal fluid (HTF) medium (Quinn et al., 1985
) supplemented with 9% (v/v) of a pasteurized human plasma protein solution (PPS) obtained from the Central Laboratory of the Blood Transfusion Service (Amsterdam, The Netherlands) and with 80 IU hyaluronidase/ml (type VIII; Sigma Chemical Co., St Louis, MO, USA, cat. no. H-3757), followed by aspiration of the oocytes in and out of hand-drawn glass pipettes. Oocytes were rinsed several times in culture medium and incubated until the injection procedure. Micro-injection pipettes used were purchased from a commercial supplier (Humagen; Gynotech, Malden, The Netherlands). ICSI was performed using hydraulic remote control joystick manipulators (Narishige; Paes Nederland BV, Zoeterwoude, The Netherlands) mounted on an Olympus IX-70 inverted microscope equipped with a Linkam heated stage (Paes Nederland BV). Just before the ICSI procedure, 1 µl of a seminal plasma-free sperm suspension was pipetted into a 5 µl droplet of a 10% polyvinylpyrrolidone (PVP) solution (MediCult; Innogenetics, Nijmegen, The Netherlands). Injection was performed in 15 µl droplets of HEPES-buffered HTF solution. All droplets were covered with embryo-tested mineral oil (Sigma, cat. no. M-8410). For ICSI, only oocytes at metaphase of the second meiotic division were used. Only motile spermatozoa were used for injection, and care was taken to select spermatozoa that were morphologically apparently normal, if available. They were immobilized by touching the tail with the micro-injection needle and injected head-first into the oocyte with the polar body at either 6 or 12 o'clock to avoid the passage of the pipette through the cytoplasmic region containing the meiotic spindle with the oocyte chromosomes. After the pipette was inserted into the oocyte, gentle suction was applied until the breakage of the oocyte membrane was observed. Care was taken to aspirate as little as possible cytoplasm into the injection pipette, and the spermatozoon was injected into the oocyte with the smallest volume of the PVP solution possible.
Culture procedures
Oocytes and embryos were cultured in 50 µl and 20 µl droplets respectively, under mineral oil. During the first half of the study period, embryos were cultured separately, in order to study development of each embryo individually in relation to features of the injection procedure. These results will be published separately. During the second half of the study period, embryos were cultured communally with a maximum of five embryos per drop.
During most of the present study period, two other studies concerning culture procedures were performed. In one study, oocytes and embryos were alternately allocated per set of two treatment cycles to culture either under ambient (20%) or reduced (5%) O2 (Dumoulin et al., 1999
). After ending this study, 5% O2 was used for all subsequent cycles. In the second study, oocytes and embryos from each consecutive treatment cycle were alternately allocated to the use of either of two culture media: a ready-to-use commercially available medium: IVF-50TM (Scandinavian IVF Science AB, Göteborg, Sweden) or `in-house' prepared HTF medium (Quinn et al., 1985
), supplemented with 9% (v/v) PPS. During a short period in the beginning of the present study period, HTF medium was used for all cycles. By this allocation procedure, a random distribution of treatment cycles over the four different combinations of culture techniques was ensured and the studies were independent.
After incubation for 1820 h, the oocytes were checked for the presence of pronuclei as proof of fertilization, washed once and, after transfer to fresh medium, cultured for another day. At the second and third day after oocyte recovery, developmental stages and morphological aspects of all embryos originating from normally fertilized oocytes were assessed under an inverted microscope with x200 magnification according to published criteria (Bolton et al., 1989). For each embryo, an embryo score was calculated by multiplying the morphological grade (values of 1 for poorest and 4 for best grade) by the number of blastomeres (Steer et al., 1992
). For each treatment cycle, the score of all embryos was averaged to obtain a mean embryo score (MES). Embryo transfer was routinely performed on day 2 after ovum retrieval, or, in a minority of the cases, on day 3 for reasons of convenience. If available, two or three embryos, depending on the developmental stage and morphological appearance of the embryos, as well as on the age of the patient, were transferred. After transfer, any supernumerary embryos were cultured until the third day after ovum retrieval. Cryopreservation of supernumerary embryos was performed on the morning of the third day if one or more embryos had reached the 8-cell stage, and if they were of good morphological quality (grades 3 and 4, Bolton et al., 1989
).
Culture of human surplus embryos
If cryopreservation was not deemed feasible, surplus embryos originating from normally fertilized oocytes were used in the present study, or in two other small studies running in our centre (Dreesen et al., 1998; Dumoulin, et al., 1998
). These studies have been approved by the local Ethics Committee. To avoid selection bias, surplus embryos of all treatment cycles during prearranged periods were used in only one study. In the present study, surplus embryos were left in their original culture medium for another 2 or 3 days. Developmental stages were recorded on each day of in-vitro development. On the morning of day 5 after ovum retrieval, surplus embryos that cavitated to form blastocyst-like structures (defined as a rim of cells surrounding a large cavity of extracellular fluid accumulated within the embryo) were fixed and stained with 4',6-diamidino-2-phenylindole (DAPI) as described earlier (Coonen et al., 1994
). The number of nuclei stained with DAPI was taken as the number of cells of the embryo. All other embryos, including those that had only just started to form a small blastocoelic cavity, were cultured for another day and were subsequently fixed on day 6 when they had developed to the full blastocyst stage. Fixation of embryos was performed only when the patients had given written consent.
Analysis of results
Data were analysed by 2 test or unpaired Student's t-test where appropriate.
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Results |
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Discussion |
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Compared with the IVF group, embryonic development in our study was more advanced in the ICSI group on the second day of development, which can be explained by the findings of Nagy et al. that the pronuclear development and cleavage to the 2-cell stage take place several hours earlier after ICSI than after IVF (Nagy et al., 1998). One day later the embryos derived from ICSI seem to have lost their head start advantage (Table II
), whereas their late preimplantation development was even impaired (Table IV
), relative to the IVF group. These results confirm in a large series of consecutive treatment cycles the earlier reports on a lower rate of blastocyst formation of embryos derived from ICSI treatment as compared with IVF. Eighty-five blastocysts out of 173 supernumerary embryos obtained in 45 cycles after IVF (47%), and 31 blastocysts out of 120 supernumerary embryos obtained in 46 cycles after ICSI (27%) have been reported (Shoukir et al., 1998
). Also several preliminary reports have been published showing lower blastulation rates of embryos obtained after ICSI (abstract by Peters and Catt, 1998; abstract by Moreno et al., 1998). Also in the cat, indications of impaired blastocyst development of embryos obtained after ICSI were found (Pope et al., 1998
).
As during the 4-year period described in this study we used several culture conditions that are known to affect human blastocyst formation, we carefully examined them for their impact on the comparison of development between IVF- and ICSI-derived embryos. It has been shown in several studies that preimplantation embryos benefit from grouped culture (Moessner and Dodson, 1995; Almagor et al., 1996
). These findings were confirmed in our study, although our data must be viewed with caution as they were collected in two different study periods. As explained earlier, it was not our intention to study the effect of group culture on embryonic development. The embryos were cultured singly in the first half of the study period in order to examine development of each embryo individually in relation to features of the injection procedure. A second culture condition that affects embryonic development in vitro is the use of a low oxygen concentration, which has been shown to be beneficial for development to the blastocyst stage in the human (Dumoulin et al., 1999
), and in various animal species, e.g. mouse, hamster, rabbit, rat, pig, sheep, cow, and goat (for references, see Dumoulin et al., 1999). The third culture condition known to affect development to the blastocyst stage is the culture medium used (Jones et al., 1998
; Martin et al., 1998
). In our study, significantly more embryos developed to the blastocyst stage when cultured in HTF medium as compared with IVF-50TM medium. It must be noted that both culture media used in the present study are probably not as suitable as the recently developed sequential media to support the development of viable blastocysts in vitro (Jones et al., 1998
). The use of these suboptimal media for blastocyst development, as well as the use of only minor quality surplus embryos (being all untransferred embryos that were considered unsuitable for cryopreservation because of their major fragmentation and/or low cell number at the third day of development) can explain the rather low number of cells per blastocyst in the present study (54 and 56 on day 6, Table VII
). The mean cell number of human blastocysts on day 6 of development has been shown to be related to culture conditions used (e.g. co-culture and type of medium) and quality of embryos included in the study, and consequently varies considerably between different reports and between different study groups (4173, Hardy et al., 1995; 42 and 87, Vlad et al., 1996; 64, Evsikov and Verlinsky, 1998; 44286, Fong and Bongso, 1998; 61 and 74, Devreker et al., 1999; 54, Jurisicova et al., 1999). However, the purpose of our study was not to achieve high blastulation rates per se, but to study differences in blastulation rates between the IVF and ICSI groups. As our experimental design ensured an equal distribution of the treatment cycles over the different culture conditions, resulting also in a comparable random distribution of surplus embryos (Table I
), the use of these different culture techniques does not influence the comparison of the blastocyst formation of surplus embryos resulting from IVF or ICSI. Furthermore, the mean incidence of blastocyst formation per cycle showed similar differences in favour of the IVF group as compared with the ICSI group in all culture conditions used.
The discrepancy between our findings in the ICSI group of on the one hand higher pregnancy and implantation rates, and on the other hand impaired embryonic development to the blastocyst stage in vitro seems hard to explain. A relationship has been suggested between poor sperm quality and embryo quality, i.e. poor embryo morphology on the second day of development (Parinaud et al., 1993), and impaired blastocyst formation (Janny and Ménézo, 1994
). It cannot be ruled out, however, that the observed effects in our studies using a conventional IVF technique were caused by a relative delay in the fertilization process which does not apply to the situation in our ICSI group in which fertilization is forced by injection. However, as discussed recently (Edwards, 1999
), the ICSI technique harbours several risks and potential dangers from different sources so that one might expect that embryonic development would be impaired. First of all, the use of spermatozoa from men with severe sperm defects must be taken into consideration. Several lines of evidence have indicated that semen from infertile men contains an increased frequency of spermatozoa with genetic defects, and it was recently shown (Twigg et al., 1998
) that such abnormal spermatozoa are well capable of forming normal pronuclei after ICSI. Spermatozoa from infertile men having a normal somatic karyotype show a slightly but significantly increased frequency of chromosomal abnormalities (Moosani et al., 1995
). A recent study (Pang et al., 1999
) reported a total aneuploidy frequency in the range of 3374% in the spermatozoa from nine randomly selected patients with oligoasthenoteratozoospermia undergoing ICSI, while the total aneuploidy in spermatozoa of controls ranged from 4.1 to 7.7%. In an earlier study (Lee et al., 1996
) it was demonstrated that the incidence of numerical and structural chromosome abnormalities in spermatozoa with highly aberrant head morphologies was about four times higher than in those with morphologically normal heads. Although we took care to select apparently morphologically normal spermatozoa, it could not be avoided in cases of extreme oligoteratozoospermia that only morphologically abnormal spermatozoa were found and injected. Other studies found higher DNA damage in spermatozoa from infertile males. It has been demonstrated (Hughes et al., 1996
) that, although the baseline level of DNA damage in normozoospermic fertile men was similar to that in asthenozoospermic infertile men, the latter group was more susceptible to damage. Also oxidative DNA damage (Kodama et al., 1997
; Twigg et al., 1998
) as well as sperm chromatin anomalies (Sakkas et al., 1996
) were found to be increased in spermatozoa of infertile male patients. Furthermore, defects of the sperm centrosome may lead to abnormal or irregular cleavage of embryos (Sathananthan, 1998
). Such defects were observed more often in asthenozoospermic sperm samples (Sathananthan, 1998
).
Besides the fact that an abnormal spermatozoon may be used for ICSI, the intracytoplasmic injection technique itself might lead to fertilization abnormalities in the zygote. It has been demonstrated in the rhesus monkey that ICSI resulted in abnormal sperm decondensation (Hewitson et al., 1999). Furthermore, these workers demonstrated the variable position of the second meiotic spindle in relation to the first polar body, which could result in damage to the spindle during the ICSI procedure as the polar body is used for microinjection targeting (Hewitson et al., 1999
).
Thus it can be postulated that the reasons for the impaired blastocyst development of embryos obtained after ICSI are that the spermatozoa used for ICSI are selected from sperm populations with relatively high incidences of fragmented DNA and chromosomal abnormalities, and/or that some oocytes are injured sublethally during the injection procedure. Both factors would result in a slightly higher incidence of embryos that would be abnormal from the start, resulting in the noted decreased incidence of blastocyst formation, but also in a higher chance of transferring a non-viable embryo into the uterus. The fact that the pregnancy and implantation rates in the ICSI group were nevertheless higher than in the IVF group can be explained by the possibly higher fertility potential of the female partners in the ICSI group as compared with those of the IVF group. Patients undergoing ICSI are selected on the basis of severe male infertility and it can be expected that most of the female partners are normally fertile. Indeed, it has been demonstrated (Le Lannou et al., 1995) that conception rates following donor insemination of women whose partners are azoospermic were significantly higher than those for women whose husbands had only moderate oligozoospermia, reflecting compromised fertility in the female partners of moderate oligozoospermic males. Furthermore, in cases of unexplained infertility, which make up 42% of our IVF group, subtle oocyte dysfunction (Hull et al., 1998
) and defective uterine receptivity (Lessey et al., 1995
) are demonstrated, leading to a reduction (Lessey et al., 1995
) are demonstrated, leading to a reduction In conclusion, we demonstrate in this study that embryos obtained after ICSI have a decreased potential to develop into blastocysts. Uncompromised fertility in the partners of severely fertility impaired men, relative to the female partners of IVF couples, appears to make up for this developmental impairment of ICSI embryos.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 10, 1999; accepted on November 5, 1999.