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Intracytoplasmic sperm injection
5 Intracytoplasmic sperm injection ANDRI~ VAN STEIRTEGHEM, ZSOLT NAGY, JIAEN LIU, HUBERT JORIS, GRETA VERHEYEN, JOHAN SMITZ, HERMAN TOURNAYE, INGEBORG LIEBAERS, PAUL DEVROEY
For more than 10 years, in-vitro fertilization (IVF) has proved to be a successful treatment to alleviate long-standing infertility in couples with tubal, idiopathic and andrological infertility. IVF may fail consistently in certain couples, especially those with severe male-factor infertility, and infrequently in couples with idiopathic infertility. A sizeable number of couples cannot be accepted for IVF because the number of motile spermatozoa present in the ejaculate is too low; for many centres the minimum number of motile spermatozoa in the ejaculate is 500000. Recent reports have indicated that these problems can be overcome by different procedures of assisted fertilization such as partial zona dissection (PZD) (Cohen et al, 1989) and subzonal insemination (SUZI) (Ng et al, 1988, 1989; Bongso et al, 1989; Fishel et al, 1990, 1991, 1992). The first pregnancies and births after replacement of embryos obtained by direct intracytoplasmic sperm injection (ICSI) into oocytes were reported by our group in the 4 July, 1992 issue of The L a n c e t (Palermo et al, 1992b). This clinical application of assisted fertilization was based on experimental work carried out on different animal species during the past few decades (Iritani, 1991). This review describes the outcome of 750 consecutive treatment cycles of assisted fertilization by SUZI and ICSI performed at the Centre for Reproductive Medicine of the Dutch-speaking Brussels Free University between October 1990 and January 1993. The outcome of these treatment cycles has previously been reported in five recent publications (Palermo et al, 1992a,b, 1993; Van Steirteghem et al, 1993a,b).
PATIENT MANAGEMENT The couples selected for these procedures of assisted fertilization by SUZI and ICSI suffered from long-standing infertility. They had undergone: (1) at least one previous IVF attempt with an adequate number of inseminated 85 Copyright© 1994,by Bailli~reTindall All rights of reproductionin any formreserved
BaiIli~re's Clinical Obstetrics and Gynaecology--
Vol. 8, No. 1, March 1994 ISBN0-7020-1844-9
86
A. V A N S T E I R T E G H E M ET A L
oocytes that had failed because oocyte fertilization had not occurred or had occurred in fewer than 5% of the oocytes; or (2) the semen parameters were too impaired for standard IVF treatment, i.e. less than 500 000 progressively motile spermatozoa were present in the total ejaculate. The procedures of assisted fertilization were reviewed and approved by the ethical committee of the University Medical Campus. The couples were fully informed about the novelty of the SUZI and ICSI procedures. After extensive patient counselling, the patients agreed and signed a consent form to have prenatal diagnosis and to be included in a prospective follow-up study of the children born after these assisted fertilization procedures.
OVARIAN STIMULATION Controlling ovarian stimulation was carried out by the association of a desensitizing protocol of the intranasally administered gonadotrophinreleasing-hormone agonist (GnRH-a) buserelin (Suprefact; Hoechst, Brussels, Belgium), human menopausal gonadotrophins (HMGs) (Pergonal; Serono, Brussels, Belgium; and Humegon; Organon, Oss, The Netherlands) and human chorionic gonadotrophins (HCGs) (Profasi; Serono; and Pregnyl; Organon). The details of this GnRH-a and H M G - H C G protocol have been reported previously (Smitz et al, 1988).
SEMEN EVALUATION Before the start of the treatment, spermatozoa selection was carried out on at least one semen sample. During the treatment cycle the male partners were initially asked for two semen samples: the first on the morning after H C G had been administered to the female partner and the second on the day of oocyte retrieval. The request for a semen sample on the day after H C G administration has recently been abandoned. Semen samples were collected by masturbation and were allowed to liquefy for at least 20 min at 37°C prior to analysis. Semen concentration and motility were assessed in a Makler counting chamber according to the World Health Organization criteria (WHO, 1992). Semen morphology was assessed by strict Kruger criteria after Diff-Quik staining (Kruger et al, 1986; Enginsu et al, 1991). Semen was considered abnormal if the sperm density was less than 20 x 106 spermatozoa per ml, if sperm motility was less than 40% and normal sperm morphology was less than 14% using strict Tygerberg criteria. The semen characteristics of the 750 consecutive cycles included: (1) 340 cycles (45%) with oligoasthenoteratozoospermia; (2) 201 cycles (27%) with two semen abnormalities; (3) 124 cycles (17%) with a single semen defect; and (4) 85 cycles (11%) with sperm density, motility and morphology within the limits of the reference ranges for semen characteristics.
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SEMEN TREATMENT
In the 28-month period of the study, several procedures were used to treat the semen to be used for SUZI and ICSI (Palermo et al, 1992a, 1993; Van Steirteghem et al, 1993a,b). For the past 9 months, the semen treatment protocol has been simplified. Seminal fluid was removed by washing the semen in a 10 ml Falcon tube with T6 medium containing 3% BSA fraction V, centrifuging at 1800g for 5 rain and removing the supernatant. The further sperm selection was done through three (90-70-50%) or two (95--47.5%) Percoll layers. The resuspended sperm pellet was put on top of the lowest Percoll gradient. After centrifugation at 300g for 20 rain, the bottom Percol layer was aspirated into a 5 ml Falcon tube. This tube was filled with T6 medium and centrifuged for 5 min at 1800g. The supernatant was removed with a Pasteur pipette and, after adding 0.2 ml T6 medium, the pellet was gently resuspended. Semen parameters were assessed after the selection procedure. The sperm suspension was then kept in the 37°C incubator (5% oxygen, 5 % carbon dioxide, 90% nitrogen) until the moment for the SUZI or ICSI procedure of the oocytes. OOCYTE COLLECTION AND PREPARATION Oocyte retrieval was carried out by vaginal ultrasound-guided puncture 36 h after HCG administration. A total of 9146 cumulus-coronoa-cell complexes were recovered and scored under the inverted microscope at x 40 and x 100 magnification. Practically all complexes were recorded as mature at the time of oocyte retrieval. The complexes were transferred into a Falcon tube with Earle's medium; these tubes were gassed prior to tight closure and were transported in a thermobox kept at 37°C to the microinjection laboratory, which is located at a distance of about 500 m. The cells of the cumulus and corona radiata were removed by incubation for about 30 s in HEPES-buffered Earle's medium with 80 IU hyaluronidase per ml (Type VIII; Sigma Chemicals, St Louis, Missouri, USA). The removal of the cumulus and corona cells was enhanced by aspiration of the complexes in and out of hand-drawn glass pipettes with an opening of about 200 lxm. Afterwards, the oocytes were rinsed several times in droplets of HEPES-buffered Earle's and B2 medium and then carefully observed under the inverted microscope at x 200 magnification. After removal of the cumulus and corona cells, 8675 (95%) oocytes revealed an intact zona pellucida and clear cytoplasm. Eighty-four per cent had extruded the first polar body, whereas 9% presented a germinal vesicle and 7% had undergone germinal vesicle breakdown. The oocytes were then incubated in 251xl microdrops of B2 medium overlayed with lightweight paraffin oil (British Drug House, Pasture, Brussels, Belgium) at 37°C in an atmosphere of 5% oxygen, 5% carbon dioxide and 90% nitrogen. About 3-4 h later, the oocytes were observed again to see whether more had extruded the first polar body. SUZI and ICSI were carried out on all morphologically intact oocytes that had extruded the first polar body.
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SUBZONAL INSEMINATION AND INTRACYTOPLASMIC SPERM INJECTION
The holding and injection pipettes were made from 30 Ixl borosilicate glass capillary tubes (Drummond Scientific, Broomall, Pennsylvania, USA). These glass capillaries were cleaned by standard procedures for tissue culture. The glass pipettes were obtained by drawing the glass capillary tubes using a horizontal microelectrode puller (Type 753; Campden Instruments, Loughborough, Leicestershire, UK). The holding pipettes were cut and fire-polished on a microforge (MF-9 Microforge; Narishige, Tokyo, Japan) to obtain an outer diameter of 60 t~m and an inner diameter of 20 txm. To prepare the injection pipette, the pulled capillary was opened on a microgrinder (EG-4 Micro-Grinder; Narishige) to an outer diameter of 7 txm and an inner diameter of 5 p~m; the bevel angle was 50°. The microforge was used to make a sharp spike on the injection pipette and to bend the edge of the holding and injection pipettes to an angle of about 45° to facilitate the injection procedure in the Petri dish. Just before the injection procedure the sperm fraction was washed. One microlitre of the sperm fraction was added to 2-4 ixl of a 1 g per 10 ml polyvinylpyrrolidone (PVP) solution in HEPES-buffered Earle's medium. A 3--5 Ixl sperm-PVP droplet was placed in the centre of a Petri dish and surrounded by eight 5 p,1 droplets of HEPES-buffered Earle's medium with 0.5 % crystalline BSA. These droplets were covered by about 3.5 ml of lightweight paraffin oil. SUZI and ICSI were carried out on the heated stage (37°C) of an inverted microscope (Diaphot; Nikon, Tokyo, Japan) at × 400 magnification. The microscope was equipped with a Nikon F-601 M camera for still pictures and a Sony video camera, allowing the procedure to be followed on a colour video monitor. The microscope was equipped with two coarse positioning manipulators and two three-dimensional hydraulic remote-control micromanipulators (Narishige). The holding and injection pipettes were fitted to a tool holder and connected by Teflon tubing to micrometer-type IM-6 microinjectors (Narishige). A single spermatozoon (for ICSI) or three to five spermatozoa (for SUZI) were selected from the central droplet and aspirated tail first into the tip of the injection pipette. For SUZI, the oocyte was held by the holding pipette, the microinjection needle introduced across the zona pellucida and three to five spermatozoa injected into the perivitelline space. For ICSI, the oocyte was held by the holding pipette with the polar body at 12 or 6 o'clock; the micropipette was pushed through the zona pellucida and into the ooplasm at 3 o'clock; and a single spermatozoon was injected into the ooplasm with approximately 1 pl medium. The injection pipette was withdrawn gently and the oocyte released from the holding pipette. FERTILIZATION AFTER SUZI AND ICSI
The further handling of the injected oocytes was similar to our standard IVF
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procedure. About 16-18 h after SUZI or ICSI, the oocytes were observed under the inverted microscope (x 200 or x 400 magnification) for any sign of damage that may have resulted from microinjection and for the presence of pronuclei and polar bodies. Fertilization was considered normal when two distinct pronuclei containing nucleoli were present. The possible presence of one pronucleus or three pronuclei was noted, together with the presence of one, two or fragmented polar bodies. The numbers of metaphase-II oocytes that survived and had two, one, or three or more pronuclei after SUZI and ICSI are summarized in Table 1. Table 1. Oocytes intact and fertilized after SUZI and ICSI.
No of metaphase-II oocytes
SUZI
ICSI
Injected Intact Pronuclear status
3287 3008 (92)*
3944 3401 (86)*
2 PN 1 PN ~>3PN
500 (17)t 161 (5)~ 62 (2)§
1873 (55)? 250 (7)~ 160 (5)§
Values in parentheses are percentages. PN, pronucleus. • ×2, 2 × 2, 1 d.f., P < 0.001 (survived versus damaged). t X2, 2 × 2, 1 d.f., P < 0.001 (number of 2 PN). $ ×2, 2 × 2, 1 d.f., P < 0.01 (number of 1 PN). §×a, 2 × 2, 1 d.f., P < 0.001 (number of 3 or more PN).
The number of oocytes that survived SUZI was higher than the number that were intact after ICSI (P<0.001). The number of normally fertilized oocytes was significantly higher after ICSI (55%) than after SUZI (17%) ( P < 0.001). More singly pronucleated oocytes were also recorded after ICSI (7%) than after SUZI (5%) (P < 0.01). The consistently higher normal fertilization rate after ICSI in comparison to SUZI led to the decision in September 1992 that all couples requiring assisted fertilization were to be treated by ICSI at the Dutch-speaking Brussels Free University Centre for Reproductive Medicine. EMBRYO CLEAVAGE AFTER SUZI AND ICSI
The embryo cleavage of the normally fertilized oocytes was evaluated after a further 24 h of in-vitro culture. The embryos were scored according to the equality of size of the blastomeres and the number of anucleate fragments (Deschact et al, 1988; Staessen et al, 1989). Cleaved embryos with less than half of their volume filled with anucleate fragments were eligible for transfer. Up to three embryos were loaded in a few microlitres of Earle's medium into a Frydman catheter and transferred into the uterine cavity. Embryo replacement was usually done about 48h after the microinjection procedure. If supernumerary embryos with fewer than 20% anucleate fragments were available, they were cryopreserved on day 2 or 3 by the slow
90
A. VAN STEIRTEGHEM ET AL Table 2. Cleavage of normally fertilized oocytes after SUZI and ICSI.
Oocytes with two pronuclei Transferable embryos Freezable embryos
SUZI
ICSI
500 400 (80)* 353 (71)t
1873 1412 (75)* 1282 (68)t
Values in parentheses are percentages. • ×2, 2 × 2 , 1 d.f., P<0.05 (transferable versus non-transferable embryos). t ×z, 2 × 2, 1 d.f., P not significant (freezable versus non-freezable embryos).
freezing protocol with dimethylsulphoxide (Van Steirteghem et al, 1987; Camus et al, 1989). The results of the further embroyo cleavage of normally fertilized oocytes was analysed after SUZI and ICSI (Table 2). The cleavage rate to transferable or freezable embryos was slightly higher after SUZI than after ICSI; these differences were significant only when transferable and non-transferable embryos were compared following SUZI or ICSI (P < 0.05). Owing to the more than threefold higher fertilization rate after ICSI, the actual number of embryos available for transfer or cryopreservation was much higher after ICSI than after SUZI. O U T C O M E OF E M B R Y O TRANSFERS AFTER SUZI A N D ICSI
The outcome of the transfer procedures is summarized in Table 3. The rate of embryo transfer in these 750 cycles was 74% (556 transfers), which is high for couples with previous fertilization failure in standard IVF or with sperm characteristics too poor to be included in standard IVF. The relative frequency of single embryo transfers was higher (54%) after SUZI, while after ICSI 56% of the replacements involved three embryos (P < 0.001). A total of 163 pregnancies were established, i.e. a pregnancy rate of 22% per cycle Table 3. Outcome of transfers after SUZI and ICSI*. SUZIt (n = 156) One embryo replaced Number (% of transfers) Pregnancies (% per transfer) Two embryos replaced Number (% of transfers) Pregnancies (% per transfer) Three embryos replaced Number (% of transfers) Pregnancies (% per transfer)
ICSIt (n = 350)
85 (54) 7 (8)
70 (20) 7 (10)
41 (26) 6 (15)
84 (24) 24 (29)
30 (19) 10 (33)
196 (56) 99 (51)
* Ten additional pregnancies were established after 50 replacements of SUZI and ICSI embryos. t P<0.001; ×2 test for percentages of cycles with one, two or three embryos replaced.
INTRACYTOPLASMIC SPERM INJECTION
91
and 29% per transfer procedure. After SUZI, 23 pregnancies were established, i.e. a pregnancy rate per transfer of 15% while after ICSI the pregnancy rate per transfer was 37% (130 pregnancies after 350 transfers). Ten additional pregnancies occurred after 50 transfers of a mixture of SUZI and ICSI embryos (i.e. a 20% pregnancy rate per transfer). As expected, the pregnancy rate was related to the number of embryos transferred. High pregnancy rates were especially prominent after triple embryo transfers following SUZI (33%) and after double (29%) and triple (51%) embryo transfers following ICSI.
EVOLUTION OF PREGNANCIES As SUZI and ICSI are novel procedures for assisted fertilization, patients were counselled to have prenatal diagnosis by chorionic villous sampling or amniocentesis. Up till now, 88 normal cytogenetic results have been recorded. Forty-five 46,XY and 43 46,XX karyotypes have been found. In one case, no fetal cell growth was observed in vitro. The safety of SUZI and especially of ICSI will require further follow-up on a larger number of treated couples who become pregnant. The evolution of the 163 pregnancies was as follows at the time of writing: (1) the 23 pregnancies after the replacement of SUZI embryos ended in 15 deliveries of 21 children; (2) the ten pregnancies after the replacement of SUZI and ICSI embryos resulted in eight deliveries of ten children; (3) the 130 pregnancies after the replacement of ICSI embryos ended prematurely in 14 preclinical and 18 clinical abortions; 98 pregnancies were evolutive; so far nine boys and 11 girls have been born following the transfer of ICSI embryos. The paediatric follow-up of the 51 children has so far revealed one major congenital malformation, i.e. the presence of cheilopalatoschisis and the duplication of renal calyces. The further results of the prospective follow-up of the children by a team of geneticists and paediatricians will allow a full-scale evaluation of safety, particularly of the ICSI procedure.
SUMMARY
Intracytoplasmic sperm injection (ICSI) is a promising assisted fertilization technique that may benefit women who have not become pregnant by invitro fertilization. ICSI and subzonal insemination (SUZI) were used to treat couples who failed fertilization after standard IVF or who could not be accepted for IVF because too few motile spermatozoa were present in the ejaculate. This paper describes the outcome of 750 consecutive cycles of SUZI and ICSI. Different aspects of these novel assisted fertilization procedures are described: patient management, ovarian stimulation, semen evaluation and treatment, oocyte collection and preparation, SUZI and ICSI techniques, assessment of fertilization and embryo cleavage, outcome of embryo transfers and the evolution of the pregnancies.
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A. VAN STEIRTEGHEM ET AL
Acknowledgements We thank the clinical, scientific, technical and nursing staff of the Centre for Reproductive Medicine for their expert assistance; Mr Frank Winter of the Language Education Centre for proofreading the text in English, and Mrs Nadia Fenners for typing the manuscript. This work was supported by grants from the Belgian Fund for Medical Research.
REFERENCES Bongso TA, Sathananthan AH, Wong PC et al (1989) Human fertilization by microinjection of immotile spermatozoa. Human Reproduction 4: 175-179. Camus M, Van den Abbeel E, Van Waesberghe L e t al (1989) Human embryo viability after freezing with dimethylsulfoxide as a cryoprotectant. Fertility and Sterility 51: 460-465. Cohen J, Malter H, Wright G e t al (1989) Partial zona dissection of human oocytes when failure of zona pellucida penetration is anticipated. Human Reproduction 4: 435-442. Deschacht J, Devroey P, Camus M e t al (1988) In-vitro fertilization with husband and donor sperm in patients with previous fertilization failures using husband sperm. Human Reproduction 3: 105-108. Enginsu ME, Dumoulin JCM, Pieters MHEC et al (1991) Evaluation of human sperm morphology using strict criteria after Diff-Quik staining: correlation of morphology with fertilization in-vitro. Human Reproduction 6: 854-858. Fishel S, Jackson P, Antinori S et al (1990) Subzonal insemination for the alleviation of infertility. Fertility and Sterility 54: 828-835. Fishel S, Antinori S, Jackson P e t al (1991) Presentation of six pregnancies established by subzonal insemination (SUZI). Human Reproduction 6: 124-130. Fishel S, Timson J, Lisi F & Rinaldi L (1992) Evaluation of 225 patients undergoing subzonal insemination for the procurement of fertilization in-vitro. Fertility and Sterility 57" 840-849. Iritani A (1991) Micromanipulation of gametes for in-vitro assisted fertilization. Molecular Reproduction and Development 28: 199-207. Kruger TF, Menkveld R, Stander FSH et al (1986) Sperm morphologic features as a prognostic factor in in-vitro fertilization. Fertility and Sterility 46: 1118--1123. Ng S-C, Bongso A, Ratnam SS et al (1988) Pregnancy after transfer of sperm under zona. Lancet ii: 790. Ng S-C, Bongso A, Chang S-I et al (1989) Transfer of human sperm into the perivitelline space of human oocytes after zona-drilling or zona-puncture. Fertility and Sterility 52: 73-78. Palermo G, Joris H, Devroey P & Van Steirteghem AC (1992a) Induction of acrosome reaction in human spermatozoa used for subzonal insemination. Human Reproduction 7: 248-254. Palermo G, Joris H, Devroey P & Van Steirteghem AC (1992b) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340: 17-18. Palermo G, Joris H, Derde M-P et al (1993) Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertility and Sterility 59: 826--835. Smitz J, Devroey P, Camus et al (1988) The luteal phase and early pregnancy after combined GnRH-agonist/HMG treatment for superovulation in IVF or GIFT. Human Reproduction 3: 585-590. Staessen C, Camus M, Khan I et al (1989) An 18-month survey of infertility treatment by invitro fertilization, gamete and zygote intrafallopian transfer, and replacement of frozenthawed embryos. Journal of in-vitro Fertilization and Embryo Transfer 6: 22-29. Van Steirteghem AC, Van den Abbeel E, Camus M et al (1987) Cryopreservation of human embryos obtained after gamete intra-fallopian transfer and/or in-vitro fertilization. Human Reproduction 2: 593-598. Van Steirteghem AC, Liu J, Joris H et al (1993a) Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. A report of a second series of 300 consecutive treatment cycles. Human Reproduction 8: 1055-1060. Van Steirteghem AC, Nagy Z, Joris et al (1993b) High fertilization and implantation rates after intracytoplasmic sperm injection. Human Reproduction 8: 1061-1066.
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World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction 3rd edn. Cambridge: Cambridge University Press.
For more than 10 years, in-vitro fertilization (IVF) has proved to be a successful treatment to alleviate long-standing infertility in couples with tubal, idiopathic and andrological infertility. IVF may fail consistently in certain couples, especially those with severe male-factor infertility, and infrequently in couples with idiopathic infertility. A sizeable number of couples cannot be accepted for IVF because the number of motile spermatozoa present in the ejaculate is too low; for many centres the minimum number of motile spermatozoa in the ejaculate is 500000. Recent reports have indicated that these problems can be overcome by different procedures of assisted fertilization such as partial zona dissection (PZD) (Cohen et al, 1989) and subzonal insemination (SUZI) (Ng et al, 1988, 1989; Bongso et al, 1989; Fishel et al, 1990, 1991, 1992). The first pregnancies and births after replacement of embryos obtained by direct intracytoplasmic sperm injection (ICSI) into oocytes were reported by our group in the 4 July, 1992 issue of The L a n c e t (Palermo et al, 1992b). This clinical application of assisted fertilization was based on experimental work carried out on different animal species during the past few decades (Iritani, 1991). This review describes the outcome of 750 consecutive treatment cycles of assisted fertilization by SUZI and ICSI performed at the Centre for Reproductive Medicine of the Dutch-speaking Brussels Free University between October 1990 and January 1993. The outcome of these treatment cycles has previously been reported in five recent publications (Palermo et al, 1992a,b, 1993; Van Steirteghem et al, 1993a,b).
PATIENT MANAGEMENT The couples selected for these procedures of assisted fertilization by SUZI and ICSI suffered from long-standing infertility. They had undergone: (1) at least one previous IVF attempt with an adequate number of inseminated 85 Copyright© 1994,by Bailli~reTindall All rights of reproductionin any formreserved
BaiIli~re's Clinical Obstetrics and Gynaecology--
Vol. 8, No. 1, March 1994 ISBN0-7020-1844-9
86
A. V A N S T E I R T E G H E M ET A L
oocytes that had failed because oocyte fertilization had not occurred or had occurred in fewer than 5% of the oocytes; or (2) the semen parameters were too impaired for standard IVF treatment, i.e. less than 500 000 progressively motile spermatozoa were present in the total ejaculate. The procedures of assisted fertilization were reviewed and approved by the ethical committee of the University Medical Campus. The couples were fully informed about the novelty of the SUZI and ICSI procedures. After extensive patient counselling, the patients agreed and signed a consent form to have prenatal diagnosis and to be included in a prospective follow-up study of the children born after these assisted fertilization procedures.
OVARIAN STIMULATION Controlling ovarian stimulation was carried out by the association of a desensitizing protocol of the intranasally administered gonadotrophinreleasing-hormone agonist (GnRH-a) buserelin (Suprefact; Hoechst, Brussels, Belgium), human menopausal gonadotrophins (HMGs) (Pergonal; Serono, Brussels, Belgium; and Humegon; Organon, Oss, The Netherlands) and human chorionic gonadotrophins (HCGs) (Profasi; Serono; and Pregnyl; Organon). The details of this GnRH-a and H M G - H C G protocol have been reported previously (Smitz et al, 1988).
SEMEN EVALUATION Before the start of the treatment, spermatozoa selection was carried out on at least one semen sample. During the treatment cycle the male partners were initially asked for two semen samples: the first on the morning after H C G had been administered to the female partner and the second on the day of oocyte retrieval. The request for a semen sample on the day after H C G administration has recently been abandoned. Semen samples were collected by masturbation and were allowed to liquefy for at least 20 min at 37°C prior to analysis. Semen concentration and motility were assessed in a Makler counting chamber according to the World Health Organization criteria (WHO, 1992). Semen morphology was assessed by strict Kruger criteria after Diff-Quik staining (Kruger et al, 1986; Enginsu et al, 1991). Semen was considered abnormal if the sperm density was less than 20 x 106 spermatozoa per ml, if sperm motility was less than 40% and normal sperm morphology was less than 14% using strict Tygerberg criteria. The semen characteristics of the 750 consecutive cycles included: (1) 340 cycles (45%) with oligoasthenoteratozoospermia; (2) 201 cycles (27%) with two semen abnormalities; (3) 124 cycles (17%) with a single semen defect; and (4) 85 cycles (11%) with sperm density, motility and morphology within the limits of the reference ranges for semen characteristics.
INTRACYTOPLASMIC SPERM INJECTION
87
SEMEN TREATMENT
In the 28-month period of the study, several procedures were used to treat the semen to be used for SUZI and ICSI (Palermo et al, 1992a, 1993; Van Steirteghem et al, 1993a,b). For the past 9 months, the semen treatment protocol has been simplified. Seminal fluid was removed by washing the semen in a 10 ml Falcon tube with T6 medium containing 3% BSA fraction V, centrifuging at 1800g for 5 rain and removing the supernatant. The further sperm selection was done through three (90-70-50%) or two (95--47.5%) Percoll layers. The resuspended sperm pellet was put on top of the lowest Percoll gradient. After centrifugation at 300g for 20 rain, the bottom Percol layer was aspirated into a 5 ml Falcon tube. This tube was filled with T6 medium and centrifuged for 5 min at 1800g. The supernatant was removed with a Pasteur pipette and, after adding 0.2 ml T6 medium, the pellet was gently resuspended. Semen parameters were assessed after the selection procedure. The sperm suspension was then kept in the 37°C incubator (5% oxygen, 5 % carbon dioxide, 90% nitrogen) until the moment for the SUZI or ICSI procedure of the oocytes. OOCYTE COLLECTION AND PREPARATION Oocyte retrieval was carried out by vaginal ultrasound-guided puncture 36 h after HCG administration. A total of 9146 cumulus-coronoa-cell complexes were recovered and scored under the inverted microscope at x 40 and x 100 magnification. Practically all complexes were recorded as mature at the time of oocyte retrieval. The complexes were transferred into a Falcon tube with Earle's medium; these tubes were gassed prior to tight closure and were transported in a thermobox kept at 37°C to the microinjection laboratory, which is located at a distance of about 500 m. The cells of the cumulus and corona radiata were removed by incubation for about 30 s in HEPES-buffered Earle's medium with 80 IU hyaluronidase per ml (Type VIII; Sigma Chemicals, St Louis, Missouri, USA). The removal of the cumulus and corona cells was enhanced by aspiration of the complexes in and out of hand-drawn glass pipettes with an opening of about 200 lxm. Afterwards, the oocytes were rinsed several times in droplets of HEPES-buffered Earle's and B2 medium and then carefully observed under the inverted microscope at x 200 magnification. After removal of the cumulus and corona cells, 8675 (95%) oocytes revealed an intact zona pellucida and clear cytoplasm. Eighty-four per cent had extruded the first polar body, whereas 9% presented a germinal vesicle and 7% had undergone germinal vesicle breakdown. The oocytes were then incubated in 251xl microdrops of B2 medium overlayed with lightweight paraffin oil (British Drug House, Pasture, Brussels, Belgium) at 37°C in an atmosphere of 5% oxygen, 5% carbon dioxide and 90% nitrogen. About 3-4 h later, the oocytes were observed again to see whether more had extruded the first polar body. SUZI and ICSI were carried out on all morphologically intact oocytes that had extruded the first polar body.
88
A. VAN STEIRTEGHEM ET AL
SUBZONAL INSEMINATION AND INTRACYTOPLASMIC SPERM INJECTION
The holding and injection pipettes were made from 30 Ixl borosilicate glass capillary tubes (Drummond Scientific, Broomall, Pennsylvania, USA). These glass capillaries were cleaned by standard procedures for tissue culture. The glass pipettes were obtained by drawing the glass capillary tubes using a horizontal microelectrode puller (Type 753; Campden Instruments, Loughborough, Leicestershire, UK). The holding pipettes were cut and fire-polished on a microforge (MF-9 Microforge; Narishige, Tokyo, Japan) to obtain an outer diameter of 60 t~m and an inner diameter of 20 txm. To prepare the injection pipette, the pulled capillary was opened on a microgrinder (EG-4 Micro-Grinder; Narishige) to an outer diameter of 7 txm and an inner diameter of 5 p~m; the bevel angle was 50°. The microforge was used to make a sharp spike on the injection pipette and to bend the edge of the holding and injection pipettes to an angle of about 45° to facilitate the injection procedure in the Petri dish. Just before the injection procedure the sperm fraction was washed. One microlitre of the sperm fraction was added to 2-4 ixl of a 1 g per 10 ml polyvinylpyrrolidone (PVP) solution in HEPES-buffered Earle's medium. A 3--5 Ixl sperm-PVP droplet was placed in the centre of a Petri dish and surrounded by eight 5 p,1 droplets of HEPES-buffered Earle's medium with 0.5 % crystalline BSA. These droplets were covered by about 3.5 ml of lightweight paraffin oil. SUZI and ICSI were carried out on the heated stage (37°C) of an inverted microscope (Diaphot; Nikon, Tokyo, Japan) at × 400 magnification. The microscope was equipped with a Nikon F-601 M camera for still pictures and a Sony video camera, allowing the procedure to be followed on a colour video monitor. The microscope was equipped with two coarse positioning manipulators and two three-dimensional hydraulic remote-control micromanipulators (Narishige). The holding and injection pipettes were fitted to a tool holder and connected by Teflon tubing to micrometer-type IM-6 microinjectors (Narishige). A single spermatozoon (for ICSI) or three to five spermatozoa (for SUZI) were selected from the central droplet and aspirated tail first into the tip of the injection pipette. For SUZI, the oocyte was held by the holding pipette, the microinjection needle introduced across the zona pellucida and three to five spermatozoa injected into the perivitelline space. For ICSI, the oocyte was held by the holding pipette with the polar body at 12 or 6 o'clock; the micropipette was pushed through the zona pellucida and into the ooplasm at 3 o'clock; and a single spermatozoon was injected into the ooplasm with approximately 1 pl medium. The injection pipette was withdrawn gently and the oocyte released from the holding pipette. FERTILIZATION AFTER SUZI AND ICSI
The further handling of the injected oocytes was similar to our standard IVF
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procedure. About 16-18 h after SUZI or ICSI, the oocytes were observed under the inverted microscope (x 200 or x 400 magnification) for any sign of damage that may have resulted from microinjection and for the presence of pronuclei and polar bodies. Fertilization was considered normal when two distinct pronuclei containing nucleoli were present. The possible presence of one pronucleus or three pronuclei was noted, together with the presence of one, two or fragmented polar bodies. The numbers of metaphase-II oocytes that survived and had two, one, or three or more pronuclei after SUZI and ICSI are summarized in Table 1. Table 1. Oocytes intact and fertilized after SUZI and ICSI.
No of metaphase-II oocytes
SUZI
ICSI
Injected Intact Pronuclear status
3287 3008 (92)*
3944 3401 (86)*
2 PN 1 PN ~>3PN
500 (17)t 161 (5)~ 62 (2)§
1873 (55)? 250 (7)~ 160 (5)§
Values in parentheses are percentages. PN, pronucleus. • ×2, 2 × 2, 1 d.f., P < 0.001 (survived versus damaged). t X2, 2 × 2, 1 d.f., P < 0.001 (number of 2 PN). $ ×2, 2 × 2, 1 d.f., P < 0.01 (number of 1 PN). §×a, 2 × 2, 1 d.f., P < 0.001 (number of 3 or more PN).
The number of oocytes that survived SUZI was higher than the number that were intact after ICSI (P<0.001). The number of normally fertilized oocytes was significantly higher after ICSI (55%) than after SUZI (17%) ( P < 0.001). More singly pronucleated oocytes were also recorded after ICSI (7%) than after SUZI (5%) (P < 0.01). The consistently higher normal fertilization rate after ICSI in comparison to SUZI led to the decision in September 1992 that all couples requiring assisted fertilization were to be treated by ICSI at the Dutch-speaking Brussels Free University Centre for Reproductive Medicine. EMBRYO CLEAVAGE AFTER SUZI AND ICSI
The embryo cleavage of the normally fertilized oocytes was evaluated after a further 24 h of in-vitro culture. The embryos were scored according to the equality of size of the blastomeres and the number of anucleate fragments (Deschact et al, 1988; Staessen et al, 1989). Cleaved embryos with less than half of their volume filled with anucleate fragments were eligible for transfer. Up to three embryos were loaded in a few microlitres of Earle's medium into a Frydman catheter and transferred into the uterine cavity. Embryo replacement was usually done about 48h after the microinjection procedure. If supernumerary embryos with fewer than 20% anucleate fragments were available, they were cryopreserved on day 2 or 3 by the slow
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A. VAN STEIRTEGHEM ET AL Table 2. Cleavage of normally fertilized oocytes after SUZI and ICSI.
Oocytes with two pronuclei Transferable embryos Freezable embryos
SUZI
ICSI
500 400 (80)* 353 (71)t
1873 1412 (75)* 1282 (68)t
Values in parentheses are percentages. • ×2, 2 × 2 , 1 d.f., P<0.05 (transferable versus non-transferable embryos). t ×z, 2 × 2, 1 d.f., P not significant (freezable versus non-freezable embryos).
freezing protocol with dimethylsulphoxide (Van Steirteghem et al, 1987; Camus et al, 1989). The results of the further embroyo cleavage of normally fertilized oocytes was analysed after SUZI and ICSI (Table 2). The cleavage rate to transferable or freezable embryos was slightly higher after SUZI than after ICSI; these differences were significant only when transferable and non-transferable embryos were compared following SUZI or ICSI (P < 0.05). Owing to the more than threefold higher fertilization rate after ICSI, the actual number of embryos available for transfer or cryopreservation was much higher after ICSI than after SUZI. O U T C O M E OF E M B R Y O TRANSFERS AFTER SUZI A N D ICSI
The outcome of the transfer procedures is summarized in Table 3. The rate of embryo transfer in these 750 cycles was 74% (556 transfers), which is high for couples with previous fertilization failure in standard IVF or with sperm characteristics too poor to be included in standard IVF. The relative frequency of single embryo transfers was higher (54%) after SUZI, while after ICSI 56% of the replacements involved three embryos (P < 0.001). A total of 163 pregnancies were established, i.e. a pregnancy rate of 22% per cycle Table 3. Outcome of transfers after SUZI and ICSI*. SUZIt (n = 156) One embryo replaced Number (% of transfers) Pregnancies (% per transfer) Two embryos replaced Number (% of transfers) Pregnancies (% per transfer) Three embryos replaced Number (% of transfers) Pregnancies (% per transfer)
ICSIt (n = 350)
85 (54) 7 (8)
70 (20) 7 (10)
41 (26) 6 (15)
84 (24) 24 (29)
30 (19) 10 (33)
196 (56) 99 (51)
* Ten additional pregnancies were established after 50 replacements of SUZI and ICSI embryos. t P<0.001; ×2 test for percentages of cycles with one, two or three embryos replaced.
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and 29% per transfer procedure. After SUZI, 23 pregnancies were established, i.e. a pregnancy rate per transfer of 15% while after ICSI the pregnancy rate per transfer was 37% (130 pregnancies after 350 transfers). Ten additional pregnancies occurred after 50 transfers of a mixture of SUZI and ICSI embryos (i.e. a 20% pregnancy rate per transfer). As expected, the pregnancy rate was related to the number of embryos transferred. High pregnancy rates were especially prominent after triple embryo transfers following SUZI (33%) and after double (29%) and triple (51%) embryo transfers following ICSI.
EVOLUTION OF PREGNANCIES As SUZI and ICSI are novel procedures for assisted fertilization, patients were counselled to have prenatal diagnosis by chorionic villous sampling or amniocentesis. Up till now, 88 normal cytogenetic results have been recorded. Forty-five 46,XY and 43 46,XX karyotypes have been found. In one case, no fetal cell growth was observed in vitro. The safety of SUZI and especially of ICSI will require further follow-up on a larger number of treated couples who become pregnant. The evolution of the 163 pregnancies was as follows at the time of writing: (1) the 23 pregnancies after the replacement of SUZI embryos ended in 15 deliveries of 21 children; (2) the ten pregnancies after the replacement of SUZI and ICSI embryos resulted in eight deliveries of ten children; (3) the 130 pregnancies after the replacement of ICSI embryos ended prematurely in 14 preclinical and 18 clinical abortions; 98 pregnancies were evolutive; so far nine boys and 11 girls have been born following the transfer of ICSI embryos. The paediatric follow-up of the 51 children has so far revealed one major congenital malformation, i.e. the presence of cheilopalatoschisis and the duplication of renal calyces. The further results of the prospective follow-up of the children by a team of geneticists and paediatricians will allow a full-scale evaluation of safety, particularly of the ICSI procedure.
SUMMARY
Intracytoplasmic sperm injection (ICSI) is a promising assisted fertilization technique that may benefit women who have not become pregnant by invitro fertilization. ICSI and subzonal insemination (SUZI) were used to treat couples who failed fertilization after standard IVF or who could not be accepted for IVF because too few motile spermatozoa were present in the ejaculate. This paper describes the outcome of 750 consecutive cycles of SUZI and ICSI. Different aspects of these novel assisted fertilization procedures are described: patient management, ovarian stimulation, semen evaluation and treatment, oocyte collection and preparation, SUZI and ICSI techniques, assessment of fertilization and embryo cleavage, outcome of embryo transfers and the evolution of the pregnancies.
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Acknowledgements We thank the clinical, scientific, technical and nursing staff of the Centre for Reproductive Medicine for their expert assistance; Mr Frank Winter of the Language Education Centre for proofreading the text in English, and Mrs Nadia Fenners for typing the manuscript. This work was supported by grants from the Belgian Fund for Medical Research.
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World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction 3rd edn. Cambridge: Cambridge University Press.