Laser-assisted ICSI: a novel approach to obtain higher oocyte survival and embryo quality rates

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1 Human Reproduction Vol.17, No.10 pp , 2002 Laser-assisted ICSI: a novel approach to obtain higher oocyte survival and embryo quality rates S.Abdelmassih 1, J.Cardoso 1, V.Abdelmassih 1, J.A.Dias 1, R.Abdelmassih 1 and Z.P.Nagy 2,3 1 Clínica e Centro de Pesquisa em Reprodução Humana Roger Abdelmassih, Rua Maestro Elias Lobo, 805, , São Paulo, SP, Brasil and 2 Reproductive Biology Associates, 1150 Lake Hearn Drive, Suite 400, Atlanta, Georgia 30342, USA 3 To whom correspondence should be addressed. nagy.zsolt.peter@iol.it BACKGROUND: Degeneration of oocytes occurs even when maximum care is exercised during ICSI, especially when the oolemma is very fragile and/or the zona pellucida is resistant. In order to be able to minimize the risk of degeneration associated with microinjection this study applied a new method: a microhole on the zona pellucida of the oocyte was drilled by laser beam just prior to ICSI to permit the penetration of the microneedle without any trauma. METHODS: A total of 32 patients (32 cycles) who had one or more previously failed ICSI cycles with a high degeneration rate of oocytes (>20%) were included in the study. Oocytes of the same patients were randomly divided into the study group [laser-assisted ICSI (LA-ICSI)] and the control group [conventional ICSI (C-ICSI)]. The outcomes of the cycles were compared and analysed. RESULTS: After LA-ICSI compared with C-ICSI, survival rates of oocytes were 99.6 and 84% (P < ), fertilization rates were 76.6 and 68.6% (not significant) and embryo development rates ( 6 cells on day 3) were 76.5 and 57.3% (P ) respectively. CONCLUSIONS: Creating a microhole on the zona pellucida of the oocyte by laser beam prior to ICSI provides a less traumatic penetration of the injection needle into the ooplasm and results in lower degeneration and higher embryo development rates than C-ICSI in patients with fragile oocytes. Key words: embryo degeneration/embryo development/fragile membrane/laser-assisted ICSI/oocyte Introduction Lanzendorf et al. reported the first normal fertilization after injection of a single spermatozoon into a human oocyte (Lanzendorf et al., 1988). It was shown that a glass needle used to deliver a spermatozoon deeply into the ooplasma could surpass the oolemma while the integrity of the oocyte was maintained. The first pregnancies obtained by ICSI were reported by Palermo et al. only 4 years after those attempts (Palermo et al., 1992). Since then, the ICSI procedure has progressively replaced previous techniques of gamete micromanipulation (Palermo et al., 1996) and today it is the treatment of choice in cases of male factor infertility and in cases of failed conventional IVF cycles. Because of the very steady success rates of ICSI a similar (or occasionally higher) fertilization rate as compared with conventional IVF and comparable embryo quality and pregnancy rates many IVF centres have extended ICSI treatment to patients other than those with male factor infertility (Aboulghar et al., 1996; Griffiths et al., 2000; Bhattacharya et al., 2001). However, it is a general experience that certain biological and technical parameters may influence the outcome of ICSI independently of the cause of the infertility. Fertilization failure after ICSI can occur due to a variety of factors (Liu et al., 1995), the most important of which are: (i) retrieval of few oocytes, and (ii) high degeneration rates after ICSI. When very few oocytes are available, degeneration of the oocytes after ICSI may result in losing the cycle because there are no embryos available for transfer. On the other hand, there are patients with a large number of retrieved oocytes that have a high degeneration rate after ICSI because of the fragility of the oocyte membrane (Nagy et al., 1995; Palermo et al., 1996). Frequently, these patients also have a poor outcome after ICSI. This is partially due to technical conditions that can be improved, although degeneration of oocytes may occur even when maximum care is exercised. This is especially true when the oolemma is very fragile and/or the zona pellucida (ZP) is very resistant (when the oocyte has to be strongly compressed in order to be penetrated by the ICSI needle) resulting in a sudden breakage of the membrane during ICSI (Nagy et al., 1995). Consequently, there is a need to develop new methods of microinjection that avoid or minimize the chance of degeneration. It has been documented that microsurgical lasers are easy to handle and allow precise working in IVF laboratories that highly facilitates its introduction in all settings (Rink et al., 1994). The introduction of laser technology seems to be a helpful tool to simplify the various techniques of gamete and embryo micromanipulation in assisted reproduction (Montag et al., 1999; Schopper et al., 1999). Because degeneration of oocytes is also related to the 2694 European Society of Human Reproduction and Embryology

2 Laser-assisted ICSI difficulty of passing the ZP with the ICSI needle, in order to minimize the risk of degeneration associated with microinjection this study applied a new method: a microhole was drilled on the ZP of the oocytes by laser beam just prior to ICSI to permit the penetration of the microneedle without any trauma. The objective of this study was to analyse prospectively the efficacy of this new technique on sibling oocytes. For this purpose, we compared the outcomes of laser-assisted ICSI (LA-ICSI) cycles and those using conventional ICSI (C-ICSI). Figure 1. Schematic representation of laser-assisted ICSI procedure (oocyte is always positioned in a way that the perivitelline space is the largest at the point of laser drilling and the innermost layer of zona pellucida is kept intact). Materials and methods Patients A total of 32 ICSI treatment cycles (from 32 patients) were included in the study, which was performed between August and December These patients were selected on the basis that they had one or more previous ICSI cycles with a high degeneration rate ( 20%) of oocytes following microinjection. None of the patients included in the present study had achieved clinical pregnancy in previous treatment cycle(s). All patients received written information about LA-ICSI and were given an opportunity to discuss this technique with the clinician before signing the relevant consent form. Semen treatment Semen samples were collected by masturbation and allowed to liquefy for ~20 min at 37 C prior to analysis. Analysis and treatment of semen samples have been reported previously (Abdelmassih et al., 1996). Controlled ovarian stimulation and oocyte preparation Patients were down-regulated with GnRH agonist (leuprolide acetate, Reliser; Serono, São Paulo, Brazil), 1 mg daily for an average of 14 days. The dose of GnRH agonist was reduced to 0.5 mg daily when the stimulation started. Controlled ovarian stimulation was carried out by administering recombinant (r)fsh (Gonal-F; Serono) in a dose of 300 IU/day for the first 5 days of stimulation. This dose was adjusted according to response parameters of the patient. Ultrasound control of follicle size and serum estradiol determination were performed daily. A total of IU/day of hcg (Profasi; Serono) was administered when at least two follicles of 18 mm of diameter were observed. Oocytes were recovered by ultrasound-guided puncture ~36 h after hcg administration. After retrieval, the oocytes were stored in an incubator at 37 C with 5% CO 2 for 2 4 h. Prior to micromanipulation, the oocytes were briefly (30 s) exposed to hyaluronidase (40 IU/ml Hyase-10X; Vitrolife Scandinavian IVF, Gothenburg, Sweden) containing medium (GAMETE 100; Vitrolife) in order to digest the matrix junctions of the cumulus cells. To enhance dispersion of corona radiata and minimize exposure to enzyme, oocytes were aspirated in and out of a plastic pipette (Mid Atlantic Diagnostics Inc., Medford, NZ, USA) with an inner diameter of 135 µm. An effort was made to remove completely the adhering corona radiata. Each oocyte was rinsed in several drops of medium to remove any remaining enzyme. Removal of the cumulus and corona cells was performed in a 50 µl droplet of GAMETE 100 medium (Vitrolife) under oil in order to maintain the temperature and to limit evaporation of the medium. Each oocyte was then examined under an inverted microscope at 200 magnification to assess the integrity and maturation stage. The latter was performed by observing the presence of the germinal vesicle, germinal vesicle breakdown (GVBD) or the first polar body Figure 2. Degeneration and fertilization rates and embryo quality after laser-assisted and conventional ICSI. indicating metaphase II (MII) stage oocytes. ICSI was performed in all MII oocytes. LA- and C-ICSI Oocytes from the same patients were divided into two groups. Randomization was performed when allocating MII oocytes into the injection dish. Oocytes were divided between the study and control groups in 2/3 (LA-ICSI) and in 1/3 (C-ICSI) proportions because preliminary studies (Nagy et al., 2001) indicated that LA-ICSI may be beneficial for fragile oocytes, and consequently the cycles with few MII oocytes would not be compromised. In group 1, oocytes were submitted to LA-ICSI that was performed in the following manner. Oocytes and sperm were placed into an injection dish as usual and a single, but immobilized spermatozoon was aspirated into the injection pipette. The oocyte to be injected was secured on the holding pipette so that the maximum possible distance was present between the inner surface of the ZP and the oolemma (perivitelline space) at the 3 o clock position (where the injection needle was supposed to penetrate into the oocyte). Using a laser beam generated by a 1480 nm diode laser (Fertilase; MTG, Medical Technology, Aldorf, Germany) a channel with small diameter (5 6 µm) was drilled with 3 5 low energy pulses always using <2 ms of pulse duration (depending on the characteristics of the ZP; Figure 1). Damage or any visible sign of oolemma reaction were avoided by ensuring that all oocytes were positioned so that the perivitelline space was the largest at the point of laser drilling and that the innermost layer of the ZP (~0.5 µm) was kept intact. The injection pipette was introduced through this channel and ICSI was performed as usual. Every oocyte that underwent laser perforation of the zona was injected immediately in sequence. In group 2, oocytes were submitted to the C-ICSI procedure as previously described (Abdelmassih et al., 1996). The type of membrane breakage during microinjection was also recorded as previously described (Nagy et al., 1995). In brief, (i) sudden breakage 2695

3 S.Abdelmassih et al. Table I. Data on the 32 cycles comparing laser-assisted ICSI (group 1) with conventional ICSI (group 2) and the results of the 48 previous cycles performed on the same 32 patients Parameters Group 1 Group 2 P-value a Previous cycles Number of injected oocytes Type of membrane breakage (%) Sudden b 10 (5) 51 (37) (41) Normal c 176 (88) 81 (59) (55) Difficult d 15 (7) 5 (4) NS 17 (4) Oocytes survived (%) 200 (99.6) 115 (84) (74) Normal fertilization rate (%) 154 (76.6) 94 (68.6) NS 281 (65) Embryo quality (%) Excellent 95 (61.6) 43 (45.8) (34) Good 23 (15) 9 (9.6) NS 42 (15) Poor 36 (23.4) 42 (44.6) (51) a Statistical comparisons were performed between group 1 and group 2 only using Fisher s exact test. b Type A: when oolemma was broken during the course of pushing the injection pipette inside the ooplasma. c Type B: when oolemma was broken after pushing the pipette deep inside the ooplasma and applying a minimal aspiration. d Type C D E: when strong aspiration and/or reposition of injection needle was required to break the membrane. NS not significant. of the membrane (type A) was noted when the oolemma was broken during the course of pushing the injection pipette inside the ooplasma (A1, when breakage occurred very much in the beginning and A2, when breakage occurred more deeply inside the ooplasma); (ii) normal breakage (type B) was noted when the oolemma was broken after pushing the pipette deep inside the ooplasma and applying a minimal aspiration; and (iii) difficult breakage (type C, D and E) was observed when strong aspiration and/or reposition of the injection needle was required to break the membrane (Nagy et al., 1995; Palermo et al., 1996). After injection, oocytes were incubated in 1.0 ml of IVF 100 medium (Vitrolife) and covered with mineral oil. Evaluation of oocyte integrity, fertilization and embryo development Oocytes were checked h after injection procedures (Nagy et al., 1998). Integrity of the cytoplasm and the number and size of pronuclei were observed. Normal fertilization was defined by the presence of two distinct pronuclei and two polar bodies. Normally fertilized oocytes were transferred to another Petri dish into droplets of 30 µl of IVF-100 medium for culturing until day 3. Cleavage of fertilized oocytes was assessed at 72 h post-injection: for each embryo the number and size of blastomeres were recorded as well as the percentage of fragmentation. Embryos were classified according to the following quality criteria on day 3: excellent quality ( 6 cells and <10% fragmentation), good quality ( 6 cells and 10 20% fragmentation) or poor quality ( 6 cells and 20% fragmentation). Microsurgical laser hatching On the morning of day 3, the embryos were scored and those selected for transfer were submitted to assisted hatching. Laser drilling was performed directly on the embryos in the original culture dish. The procedure used for ZP microdrilling was similar to that described in detail elsewhere (Rink et al., 1996). The drilled hole was µm wide. Embryos were positioned so that there was a clear space between the blastomeres and the ZP to ensure that the cytoplasm was not touched by the laser. If it was possible to identify the hole of the laser drilling from the first day (LA-ICSI) then laser hatching was performed at the same location on the ZP. After finishing the procedure, the dish was placed back into the incubator until the transfer procedure. Embryo transfer and luteal phase support For embryo transfer, a Edwards Wallace 23 cm catheter (SIMS Portex Ltd, Kent, UK) connected to an insulin syringe was used. The catheter was rinsed with transfer medium (IVF-50; Scandinavian IVF Science, Gothenburg, Sweden), then embryos were loaded and the catheter was handed to the clinician who inserted it through the endocervical canal. The insertion of the catheter and the position in which the embryos were deposited (1 cm from the fundus of the uterine cavity) was visualized in all cases. Contact with the fundus was avoided. Progesterone oral administration started on the day of the retrieval (800 mg daily Utrogestan; Besins Laboratories, Paris, France) and was continued until the assessment of pregnancy (12 days after transfer). Cephalexin was administered (500 mg every 6 h for 5 days) starting on the day of retrieval. The β-hcg serum levels were assessed on day 12 after transfer. A clinical pregnancy was defined as the visualization of a heartbeat by transvaginal ultrasound at 6 weeks gestation. Statistical analyses One-way analysis of variance, χ 2 -test and Fisher s exact test were applied whenever appropriate for statistical analysis. Results were considered to be significant when P Results The mean age SD of the women in these 32 cycles was years. The mean total dose of rfsh was IU/cycle. The mean number of days of rfsh administration was Mean values of estradiol and progesterone on the day of administration of hcg were pg/ml and ng/ml respectively. Mean of retrieved oocytes was per patient. A total of 415 oocytes were recovered and 338 were at the MII stage of nuclear maturity (81.4%). The number of injected oocytes was 338, from which 201 were submitted to LA-ICSI and 137 to the C-ICSI. Sudden breakage of the oocyte membrane was significantly lower in the LA-ICSI group than in the C-ICSI group (5.0 versus 37.5%; P , Table I). Survival rates of injected oocytes were significantly higher in the LA-ICSI 2696

4 Laser-assisted ICSI Table II. Data on the 32 cycles comparing laser-assisted ICSI (group 1) with conventional ICSI (group 2) with regard to embryo fragmentation and development on day 3 Parameters Group 1 Group 2 P-value Embryo development (all embryos) Embryos with 4 cells on day 3 (%) 19 (12.3) 28 (29.8) NS Embryos with 5 cells on day 3 (%) 17 (11) 12 (12.8) NS Embryos with 6 7 cells day 3 (%) 50 (32.4) 25 (26.6) NS Embryos with 8 cells day 3 (%) 68 (44.1) 29 (30.9) NS Embryos with 6 cells on day 3 (%) 118 (76.6) 54 (57.4) Fragmentation (%) 10% 95 (61.7) 43 (45.7) % 39 (25.3) 29 (30.9) NS 20% 20 (13.0) 22 (23.4) NS NS not significant. group compared with C-ICSI group (P ; Figure 2); the normal fertilization rate was not significantly higher in the LA-ICSI group (when calculated on the number of oocytes injected; P 0.13, not significant). Abnormal fertilization rates were similar both in the LA-ICSI (3.4%) and in the C-ICSI group (4.4%). The percentage of excellent quality embryos was significantly higher in the LA-ICSI compared with the C-ICSI group (P , Table I). The percentage of embryos with 10% of fragmentation (61.7 versus 45.7%; P , χ 2 -test) and the percentage of embryos with 6 cells on day 3 (76.6 versus 57.4%; P , χ 2 -test) were significantly higher in the LA-ICSI group than in the C-ICSI group respectively (Table II). A total of 110 excellent quality embryos were transferred to the patients ( per patient on average; 76 from the LA-ICSI group and 34 from the C-ICSI group). Fourteen patients had a positive serum β-hcg dosage on day 12 after transfer (44%; transferring 35 embryos from the LA-ICSI group and 14 embryos from the C-ICSI group). Twelve clinical pregnancies were obtained (six singletons and six multiples) as demonstrated by the presence of fetal heartbeats on week 6 of gestation (37.5% per cycle). An overall implantation rate of 18% was achieved in these cycles. Discussion The results of this study show that it is possible to create a microhole in the ZP of the oocyte by laser beam prior to ICSI to assist a less traumatic penetration of the injection needle into the ooplasm. Such a procedure seems to be important in cases of very fragile oocytes, for instance those with sudden breakage of the oolemma during C-ICSI. Laser technology seems to be a promising tool that has been introduced in assisted reproduction treatment in recent years. Lasers have been applied in assisted reproduction with the purpose of manipulation of the ZP of embryos to perform assisted hatching (Strohmer and Feichtinger, 1992; Antinori et al., 1996; Mantoudis et al., 2001). Assisted hatching of human embryos was described more than a decade ago (Cohen et al., 1988) and initially acid Tyrode s solution was used (Cohen et al., 1990) to facilitate the evacuation of the embryo from the ZP, which may be hardened during the in-vitro culture period (De Vos and Van Steirteghem, 2000). So far there has not been a consensus about any benefits of such procedures (Cohen et al., 1992; Lanzendorf et al., 1998; Magli et al., 1998). Lasers can also be used for preimplantation genetic diagnosis. Microdissection of the ZP with a laser system simplifies subsequent polar body biopsy or removal of blastomeres because of the advantage of using a single needle for the procedure (Veiga et al., 1997; Boada et al., 1998; Montag et al., 1998). However, other possible laser applications, such as partial zona dissection (Strohmer and Feichtinger, 1992), subzonal insemination (Schutze et al., 1994), optical tweezer sperm micromanipulation (Tadir et al., 1989) or the hemizona assay (Montag et al., 1999; Schopper et al., 1999) are not routinely used for assisted reproduction. There are different laser systems, including: (i) the contact type, for instance the erbium-yttrium-aluminium-garnet (Er:YAG) laser system which operates in the infrared region of the light spectrum (Obruca et al., 1997); and (ii) the noncontact type, which emits at a different wave length and power, for instance the holmium: yttrium scandium gallium garnet (Ho:YSGG) laser system (Neev et al., 1995), non-contact UV laser nm (Liow et al., 1996) and the 1480 nm diode laser (Germond et al., 1996; Rink et al., 1996; Montag et al., 1998; Blake et al., 2001). It has been demonstrated that the 1480 nm diode laser works without physically touching the cells, has no detectable detrimental effects (Chatzimeletiou et al., 2001) on living cells (especially if it is used with pulse duration 5 ms and laser power ~100 mw) and is easy to handle (Montag et al., 1999; Schopper et al., 1999; Douglas- Hamilton and Conia, 2001). The question of safety is always an important point when introducing a new technique. In this case, no animal experiment was performed prior to this study. However, the following points convinced us to apply LA-ICSI directly in human IVF. (i) There are extensive literature on the safe use of lasers both in animals and humans (including several normal pregnancies, as detailed above). (ii) The technical parameters of the Fertilase laser system that we used are not associated with mutagenic effects (because it is an infrared laser that emits waves that are not absorbed by nucleotides). However, the laser beam does generate a heat effect that can indeed damage cells 2697

5 S.Abdelmassih et al. depending on the energy level/pulse duration and the distance between the center of the laser beam and the cell membrane. (iii) The energy level/pulse duration used in our study was very low (5 8 times lower than that used for assisted hatching) and even if the oocytes were somewhat more sensitive than embryonic cells to the laser beam it should not cause a significant injury especially because a maximum distance was maintained in the perivitelline space. (iv) The most recently published case reports on LA-ICSI (Nagy et al., 2001; Rienzi et al., 2001) did not indicate any damaging effect in humans. However, to ensure the maximum safety at all times the greatest care was exercised during the procedure. For instance, great attention was always paid so that the distance between the perivitelline space and oolemma was the maximum at the point of laser drilling. In addition, the innermost layer of the ZP (~0.5 µm) was kept intact and a very short pulse duration ( 2 ms) was applied, which prevented any visible sign of oolemma reaction even at the last laser shot when performing LA-ICSI. As a consequence, the significantly reduced damage rate of injected oocytes and improved embryonic development in the present study may also confirm that laser-mediated ICSI is a much more gentle approach with which to inject fragile oocytes compared with C-ICSI. The high precision of the Fertilase laser system enabled us to create a channel on the ZP with a very narrow opening just wide enough to permit the passage of the ICSI needle. It was observed in mice that the diameter of the channel on the ZP has paramount importance for hatching process (Malter and Cohen, 1989), especially if it is not large enough to permit evacuation of the embryos from the ZP. A small hole may strangulate the embryo and may be a phenomenon probably associated with increased incidence of monozygotic twinning (Nijs et al., 1993; da Costa et al., 2001). Because of this fact, we made an effort to drill the smallest possible hole that permitted the passage of the ICSI needle. In the present study, the concern related to the small size of the hole was diminished in part because we additionally performed assisted hatching on all embryos selected for transfer, which resulted in a large enough opening on the ZP. However, it is not possible to completely exclude the possibility that some of the transferred embryos had two holes on the ZP. This may carry a risk that at the hatching process the embryo is escaping through two different holes and/or gets trapped in the smaller hole, which may result in twinning. In our series, we did not observe monozygotic twinning among the pregnant patients; however, this should be confirmed on more cases. We exercised maximal care during the injection procedure without compromising the integrity of the oolemma and without compression of the oocytes. When ICSI was performed in this way, the degeneration rate of oocytes was minimal and consequently the survival rate was very high, as demonstrated by our study. Although total fertilization failure is not frequent after ICSI it does still occur (Liu et al., 1995) and often it is caused by the complete or nearly complete degeneration of all oocytes. This can also happen when a patient has a relatively large number of oocytes, but it is more apparent when there are only few oocytes available. However, a much larger number of patients experience only a partial degeneration of oocytes 2698 which may not lead directly to cancellation of the cycle, but does impair the chances of pregnancy by lowering the number of survived oocytes, thus lowering the number of zygotes and embryos and consequently reducing the possibility of embryo selection for transfer. In this study, after C-ICSI a high proportion of the oocytes (37%) had a sudden breakage of the membrane, and most of them degenerated. In all of these cases the oolemma was very fragile because it was already broken at the beginning of the introduction of the injection pipette (type A1 breakage) (Nagy et al., 1995). As a consequence, a lower number of oocytes survived. Embryo development following C-ICSI was relatively poor and most had unequal sized blastomeres with much fragmentation. Performing C-ICSI on more sensitive oocytes may therefore cause a stronger trauma or sublethal damage affecting the cytoskeleton and other structures of the oocytes that can be avoided by LA-ICSI. This might explain why the developmental speed and morphological quality of embryos was significantly better after LA-ICSI than after C-ICSI in our study. Thus, such an improvement in cleavage and quality of the embryos may be related to the fact that oocytes in the LA-ICSI group were not compressed at all during microinjection, which probably resulted in a better preserved microfilament structure that is associated with cell division. Despite the fact that all of the patients in our study had fragile oocytes, a reasonably high clinical pregnancy rate of 37.5% per cycle was obtained with an implantation rate of 18%. From our viewpoint several oocytes were rescued from degeneration, which yielded a larger number of excellent quality embryos to transfer. A larger number of embryos available provided the possibility for an improved embryo selection for transfer (mainly from the LA-ICSI group) and resulted in satisfactory implantation and pregnancy rates. Importantly, the number of clinical pregnancies obtained may also indicate that our novel approach of LA-ICSI does not impair the implantation potential of the derived embryos. As a conclusion we may say that LA-ICSI is a safe, efficient, simple and easy procedure to use in the daily routine. It may prevent damage to the oocyte as demonstrated by a nearly 100% survival rate in the group of patients with a history of a high degeneration rate in previous ICSI cycle(s). It may also decrease damage to the subcellular components of oocytes, as demonstrated by better cleavage and development of embryos from the LA-ICSI group, which leads to a better yield of high quality embryos permitting improved selection for transfer and consequently to a better outcome of the cycles. Because the LA-ICSI procedure is simple and easy, it is possible to use it routinely for those patients who have a higher risk for oocyte degeneration due to oocytes with difficult ZP penetration or/ and fragile oolemma. References Abdelmassih, R., Sollia, S., Moretto, M. and Acosta, A.A. (1996) Female age is an important parameter to predict treatment outcome in intracytoplasmic sperm injection. Fertil.. Steril., 65, Aboulghar, M.A., Mansour, R.T., Serour, G.I., Amin, Y.M. and Kamal, A. (1996) Prospective controlled randomized study of in vitro fertilization versus intracytoplasmic sperm injection in the treatment of tubal factor infertility with normal semen parameters. Fertil. Steril., 66,

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