Subzonal sperm microinjection in cases of severe male factor infertility and repeated in vitro fertilization failure*

Transcription

1 FERTILITY AND STERILITY Copyright 1992 The American Fertility Society Vol. 57, No.6, June 1992 Printed on acid-free paper in U.S.A. Subzonal sperm microinjection in cases of severe male factor infertility and repeated in vitro fertilization failure* Denny Sakkas, Ph.D. t:j: Orly Lacham, M.Sc. t Luca Gianaroli, M.D. Alan Trounson, Ph.D.tll Centre for Early Human Development, Monash University, Melbourne, Australia, and IVF Centre, Casa di Cura Villa Regina, Bologna, Italy Objective: To examine (1) fertilization rates obtained with subzonal sperm microinjection when different numbers of sperm are injected into the perivitelline space; (2) when subzonal sperm microinjection is combined with dilute insemination; and (3) the association of semen quality characteristics with fertilization. Design: Subzonal sperm microinjection and subzonal sperm micro injection combined with dilute insemination was performed in 109 and 41 cycles on patients in two clinical trials in Melbourne, Australia, and Bologna, Italy, respectively. Patient Participants: Couples who have experienced repeated in vitro fertilization failure or in whom the husband has severe male factor infertility. Primary Outcome Measures: The number of oocytes fertilized after injection of different numbers of sperm into the perivitelline space, the number of patients transferred, and pregnancy outcome. Results: The injection of multiple numbers of sperm into the perivitelline space failed to improve monospermic fertilization rates but caused an increase in polyspermic fertilization. In patients with initial semen parameters exhibiting >50% motility or >50% normal morphology fertilization rates were improved when subzonal sperm microinjection-treated eggs were incubated in a dilute insemination medium. Six pregnancies were obtained, two of which have progressed to term. Conclusions: When applied to male factor patients, the sub zonal sperm micro injection technique results in a 14% to 15% fertilization rate. However, of the 102 embryos transferred only three (2.9%) fetal heart beats were obtained. Fertil Steril 1992;57: Key Words: Male infertility, sperm microinjection; fertilization; polyspermy The technique for micro injection of sperm into the perivitelline space of the mature human oocyte Received August 8, 1991; revised and accepted February 14, Supported in part by funds from the National Health and Medical Research Council of Australia, Melbourne, Victoria, Australia, as a project grant to Alan Trounson, Ph.D. t Centre for Early Human Development, Monash University. :/: Present address: Policlinique de Sterilite, Hopital Cantonal Universite de GenElVe, Geneve, Switzerland. IVF Centre, Casa di Cura Villa Regina. II Reprint requests: Alan Trounson, Ph.D., Centre for Early Human Development, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. (subzonal sperm microinjection) was developed (1-3) to try to improve the occurrence of fertilization in couples with severe male factor infertility in whom conventional in vitro fertilization (IVF) techniques were unsuccessful. Other methods have also been developed for sperm to have direct access to the oocyte cell without the necessity of entering the glycoprotein barrier during insemination in vitro. The zona pellucida may be mechanically torn with a glass needle or cracked by piercing the zona on a glassholding pipette and is termed partial zona disection (4) or the zona partially dissolved by squirting an acidified solution on the zona through a finely drawn glass micropipette and is termed zona drilling (5). Vol. 57, No.6, June 1992 Sakkas et al. Subzonal sperm micro injection 1279

2 All these procedures rely on the increased probability that a sperm capable of fertilization will come in direct contact with the oolema of the oocyte by avoiding the necessity to make their way through the zona pellucida. If numerous sperm are microinjected into the perivitelline space or the zona is disrupted before insemination, it may be difficult to control polyspermic fertilization because an intact zona pellucida is required to effectively block polyspermy (6). The use of micromanipulation techniques to obtain fertilization with sperm samples that are not capable of fertilizing zona intact oocytes raised our own concerns about increased genetic abnormalities in the resulting embryos. However, we have shown that the rate of chromosomal abnormalities in 00- cytes fertilized after subzonal sperm microinjection with very poor quality sperm samples from infertile men who were unable to fertilize their wives' oocytes in vivo or in vitro was not increased above that of oocytes fertilized by conventional IVF procedures (7). In addition, pregnancies and births have been reported for subzonal sperm microinjection (2, 3) and for partial zona disection (8, 9), even though success for zona drilling has been elusive (4, 5). These observations provided us with the confidence to initiate clinical studies of subzonal sperm microinjection for the treatment of male factor patients in IVF. This report gives the results of clinical trials undertaken in two IVF programs, examining (1) fertilization rates obtained with subzonal sperm microinjection when different numbers of sperm are injected into the perivitelline space; (2) when subzonal sperm microinjection is combined with dilute insemination; and (3) the association of semen quality characteristics with fertilization. Patients MATERIALS AND METHODS Mature human oocytes were obtained from patients undergoing IVF for the treatment of infertility at the Infertility Medical Center, Epworth Hospital, Richmond, Victoria, Australia. The results reported in this study deal with the treatment of a total of 100 patients, comprising 109 treatment cycles, between September 1989 and July In a second trial, 39 patients (41 treatment cycles) were also treated at the Casa di Cura, Villa Regina, Bologna, Italy, between January 1990 and July Infertile couples involved in the studies were either those who 1280 Sakkas et a1. Subzonal sperm microinjection had failed to produce any fertilized oocytes in two or more attempts at IVF (Melbourne 17 cases and Bologna 3 cases) or those in whom the male partner had such severely reduced semen quality after swimup or Percoll was performed «0.1 X 10 6 /ml and <60% progressive and forward motility) that conventional IVF was not considered possible. In 9 cases, couples undergoing routine IVF in Melbourne, in whom the husband had normal semen parameters, donated their excess eggs for microinjection. Preparation of Oocytes Oocytes were aspirated from the ovarian follicles shortly before the estimated time of ovulation. Superovulation was induced with clomiphene citrate (Clomid; Merrel Dow, Melbourne, Victoria, Australia) and human menopausal gonadotropin (Humegon; Organon Pty. Ltd., Lane Cove, New South Wales, Australia), and the time of oocyte recovery was timed by the injection of 5,000 IV human chorionic gonadotropin (Profasi; Serono, French's Forest, New South Wales, Australia) as previously described (10). In the Bologna trial, patients were superovulated using the protocol described by Gianaroli et al. (11). Oocytes were recovered by ultrasound-guided follicular aspiration and the oocyte cumulus complexes cultured in human tubal fluid medium (12) containing 10% maternal human serum (HS) for 2 to 6 hours at 37 C in an atmosphere of 5% CO 2 in air. The cumulus oophorous was dissolved by a brief incubation in organically buffered human tubal fluid containing hyaluronidase (3 IV/mL, Leech Type X; Sigma Chemical Co., St. Louis, MO), and the corona radiata cells were removed by gentle pipetting. Mature metaphase II oocytes with a clearly visible first polar body were microinjected after the initial 2 to 6 hour incubation after the recovery from the ovary. Those oocytes without a polar body or immature oocytes with a germinal vesicle were cultured in human tubal fluid + 10% HS for 12 to 36 hours and those that had matured to metaphase II oocytes were then microinjected with sperm. In the Bologna trial, both metaphase II oocytes and those in which the polar body had not yet been extruded were microinjected immediately after the 2 to 6-hour incubation. Preparation of Sperm Semen samples were incubated for 20 minutes at 37 C with 3 mm 2-deoxyadenosine (13) (Sigma Chemical Co.) to increase the recovery of motile sperm. The 2-deoxyadenosine was washed out dur- Fertility and Sterility

3 ing sperm preparation. Motile sperm were concentrated by either density gradient centrifugation (1) or the conventional semen-pelleting procedure used for IVF, which involved an initial 2:1 dilution of semen with human tubal fluid + 10% HS, gentle centrifugation to pellet the sperm, and swim-up into 0.5 ml of overlayered culture medium. Sperm were used for micro injection within 7 hours after preparation, and only motile sperm with grossly normal morphology were chosen from the diluted sample in the microinjection chamber. Sperm that possessed pin heads, two tails, and abnormally large head regions were avoided when selecting sperm to inject. Microinjection Oocytes were handled and microinjected using the techniques described in detail previously (1,14), using Leitz micromanipulators (Ernst Leitz, Wetzlar, Germany) and inverted microscopy at 150X magnification. In the Melbourne trial, the inverted microscope was also fitted with a warming stage to avoid cooling of the gamete during micromanipulation. For microinjection, oocytes were held stationary on a suction pipette polished by the heated filament of a de Fonbrune micro forge (Beaudouin, Paris, France), leaving an opening of 15 ~m. Sperm microinjection pipettes were made from thin walled capillaries, reduced on a micropipette puller (David Kopf Instruments, Tujunga, CA), and the opening ground to 45 (diameter 8 to 12 ~m) using a Groschopp grinder (Groschopp Co., Viersen, Germany). Microinjection was carried out in oil-filled chambers described previously by Mann (14). Cumulusdenuded oocytes were transferred from the culture medium into a droplet of organically buffered (Hepes, N -2 -hydroxyethylpiper-azine-n 1-2 -ethane sulfonic acid; Calbiochem Corp., La Jolla, CA) human tubal fluid + 10% HS in the micro injection chamber. A second droplet in the chamber contained a dilute suspension of sperm in human tubal fluid + 10% HS. One to 10 sperm were gently aspirated into the microinjection pipette, moved to the droplet containing the oocyte, and the sperm gently injected into the perivitelline space after piercing the zona pellucida. The oocytes remained in the injection chamber for a period of 2 to 3 minutes. Once the oocyte had been micro injected, it was transferred to human tubal fluid + 10% HS for culture at 37 C in an atmosphere of 5% CO 2 in air. In the Melbourne trial, microinjected eggs were placed back into Nunclon 4 well dishes containing 0.8 ml of culture medium, whereas Nunclon 35-mm Petri dishes (A/S Nunc, Roskilde, Denmark) containing 2.5 ml of medium were used in the Bologna trial. In the course of the study, three patient groups evolved; group I in which sub zonal sperm microinjection was performed and the eggs placed back in culture medium alone; group II in which subzonal sperm microinjection was performed, and one-half of the patient's eggs was placed back in culture medium alone and one-half was incubated in a dilute insemination suspension of the remainder of the patient's sperm suspension (subzonal sperm microinjection plus insemination); or group III in which sub zonal sperm micro injection plus insemination was performed on all the patients' eggs. The dilute insemination suspensions contained between 500 and 4,000 sperm/ml in cases in which the male had oligozoospermia or oligoasthenozoospermia. In cases of previous failed fertilizations, between 5,000 and 10,000 sperm/ml were present in the dilute insemination suspension. The microinjected oocytes were examined 16 to 20 hours after injection for the presence of pronuclei; those that exhibited pronuclei after 20 hours after injection were considered delayed and were not included in the overall total of fertilization. Oocytes with two distinct pronuclei (2 PN) and two polar bodies were considered fertilized. Embryo Transfer Fertilized pronuclear one-cell zygotes were transferred by the zygote intrafallopian transfer (ZIFT) procedure 22 to 24 hours after injection, and early cleavage stage embryos were transferred to the fallopian tube (tubal embryo stage transfer) by laparoscopy 30 to 40 hours after micro injection or transferred to the uterine cavity 40 to 56 hours after microinjection using conventional embryo transfer (ET) procedures (15). In the Bologna trial, the majority of patients (16/24) had fertilized one-cell 00- cytes transferred by ZIFT, whereas in the Melbourne trial the majority of patients (19/34) had embryos transferred to the uterus. Microinjection Studies In the course of the clinical trial, we adopted three main investigations ofthe microinjection technique and assessed their affect on the overall fertilization rate after microinjection. They were as follows: (1) fertilization rates obtained with subzonal sperm microinjection when different numbers of sperm are injected into the perivitelline space; (2) when subzonal sperm micro injection is combined with dilute Vol. 57, No.6, June 1992 Sakkas et al. Subzonal sperm microinjection 1281

4 insemination; and (3) the association of semen quality characteristics with fertilization. Number of Sperm Microinjected Into the Perivitelline Space In the Melbourne trial, group I and II patients' eggs were injected with between 1 and 10 sperm into the perivitelline space and incubated in medium alone. The effect on polyspermic fertilization and monospermic fertilization was compared after microinjection of varying numbers of sperm. The majority of eggs had either 1 or 4 sperm injected under the perivitelline space. In the Bologna trial, the majority of eggs had 5 or less sperm injected into the perivitelline space and were placed in the same Petri dish after microinjection; hence, a correlation of number of sperm injected into the perivitelline space and fertilization could not be performed. Subzonal Sperm Microinjection Compared With Subzonal Sperm Microinjection Plus Insemination In this study, patients' eggs were microinjected with sperm and then randomly allocated to either culture medium alone or into a dilute insemination suspension in the remainder of the patients' sperm suspension (subzonal sperm microinjection plus insemination). In this trial, group II patients' eggs were allocated to both subzonal sperm microinjection and subzonal sperm microinjection plus insemination and the fertilization rates compared. In the Melbourne trial, 13 of the 35 patients also had some eggs allocated to subzonal sperm microinjection without placing any sperm into the perivitelline space plus insemination, to investigate whether the subzonal sperm microinjection plus insemination technique could improve fertilization by allowing sperm to swim through the hole created by the microinjection pipette. The group III patients only had subzonal sperm microinjection plus insemination. Semen Quality Parameters and Fertilization Rate In an attempt to correlate fertilization results with semen characteristics, a retrospective analysis was carried out to ascertain whether specific male factor patients were more likely to achieve fertilization compared with others. Count and motility were performed on the day of microinjection. Because there was insufficient sperm to perform analysis of sperm morphology on the day of treatment, the patient's previous sperm sample was taken as an indication of the percentage of abnormal sperm present. The correlations between normal morphology and fer- tilization are therefore subject to variations occurring between different semen samples from the same patient. Several patients did not have their sperm analyzed for motility, and a greater number could not be analyzed for morphology because of severe oligozoospermia. The fertilization results obtained after treatment of 10 patients with sperm antibodies are also reported. Subzonal Sperm Microinjection Compared With IVF In the Bologna trial, 8 of the 39 couples' semen parameters were borderline on the day of treatment because the husbands' sperm number and motility were variable, and the eggs were allocated to both subzonal sperm microinjection and routine IVF. In such cases of IVF, the eggs were pooled and placed in a 5-mL tube containing the insemination medium. Comparison of the different fertilization rates were performed by the X2 test. RESULTS The outcome of all the eggs treated in both trials is summarized in Table 1. In the Melbourne trial, of the eggs that exhibited 2 PN by 20 hours after injection, 10 of 72 (13.9%) failed to cleave while the mean (±SEM) number of cells of 57 embryos assessed at 48 hours after injection was 3.5 ± 0.2. In the 109 treatment cycles, fertilization was achieved in 42 cycles while patients only received transfer of embryos in 34 cycles (31.2%). The majority of patients only received 1 or 2 embryos (Table 2). In the Bologna trial, of the 41 treatment cycles, 24 (58.5%) resulted in transfer (Table 2). Two preg- Table 1 Overall Number of Eggs Treated, Fertilized, and Transferred After Subzonal Sperm Microinjection in Both the Melbourne and Bologna Trials Melbourne Bologna No. of patients No. of cycles Total no. of eggs microinjected No. of eggs injected per cycle (mean±sem) 5.8 ± ± 0.8 Total no. of eggs fertilized by 20 hours after injection 89 (14.1)* 45 (14.6) 2PN 72 (SO.9) 43 (95.6) Polyspermic 17 (19.1) 2 (4.4) No. of 2 PN per cycle (mean ± SEM) 0.7 ± ± 0.2 Total no. of Delayed fertilization 10 (1.6) 10 (3.2) Activated 34 (5.4) 2 (0.6) * Values in parentheses are percents Sakkas et al. Subzonal sperm microinjection Fertility and Sterility

5 Table 2 Number of Patients Receiving Transfer of Fertilized Microinjected Embryos in the Melbourne Trial by Either TEST or Routine Uterine Transfer and in the Bologna Trial by ZIFT or Routine ET Melbourne Bologna * No. of embryos Uterine Uterine transferred TESTt transfer ZIFT transfer 4 1* f * Five delayed fertilized eggs were included in the transfers. t TEST, tubal embryo stage transfer. * Two zygotes replaced by ZIFT and two embryos replaced into the uterus the following day. Two singleton pregnancies delivered (both normal females). II Biochemical pregnancy. ~ Aborted in the 3rd month. nancies from the Bologna trial have progressed to term (Table 2). The two deliveries resulted from one oligoasthenozoospermic (3 X 10 6 sperm/ml and 30% total motility; 0.1 X 10 6 sperm/ml, 40% total motility after preparation) patient who had fertilization from both subzonal sperm microinjection and subzonal sperm microinjection plus insemination and an asthenozoospermic (25 X 10 6 sperm/ml and 5% progressive motility; 1.0 X 10 5 sperm/ml, 70% total motility after preparation) patient who had fertilization from subzonal sperm microinjection only. The aborted pregnancy in Melbourne was from a failed fertilization patient, whereas the biochemical pregnancies were all from oligoasthenozoospermia patients. Immature oocytes that had matured from the germinal vesicle or the metaphase I stage had only 1 of 10 (10.0%) and 1 of 30 (3.3%) eggs fertilized, respectively. The egg fertilized after maturation from the germinal vesicle stage contained 4 PN. Number of Sperm Microinjected Into the Perivitelline Space In this analysis of the Melbourne trial, all the eggs treated by subzonal sperm microinjection alone from patient groups I and II were assessed. Eggs injected with 1 or 4 sperm and incubated in medium alone had 6.9% and 13.8% fertilization rates, respectively (Fig. 1); however, this failed to reach significance (x 2 = 2.82; 0.1 > P> 0.05). The level of significant difference was further reduced because 5 of 27 (18.5%) of the fertilized eggs were polyspermic when 4 sperm were placed into the perivitelline space. When between 5 and 10 sperm were microin- jected, the total fertilization rate was not increased above that of 4 sperm, but an increase in the amount of polyspermy was evident (Fig. 1). In the Bologna trial, an overall mean (±SEM) of 3.7 ± 0.1 sperm were injected into the perivitelline space resulting in an overall fertilization rate of 45 of 309 (14.6%), of which only 2 eggs were polyspermic. Subzonal Sperm Microinjection Compared With Subzonal Sperm Microinjection Plus Insemination In an attempt to increase fertilization rates after subzonal sperm microinjection, we examined whether incubation in a dilute sperm suspension after subzonal sperm microinjection increased fertilization (subzonal sperm microinjection plus insemination). The fertilization results of the patients treated in the Melbourne and Bologna trials undergoing sub zonal sperm microinjection only, subzonal sperm microinjection and subzonal sperm microinjection plus insemination, or subzonal sperm microinjection plus insemination alone are summarized in Table 3. Of the 35 patients whose eggs were allocated to both subzonal sperm microinjection and subzonal sperm microinjection plus insemination in the Melbourne trial, there was an increase in the number of eggs fertilized and displaying 2 PN when using subzonal sperm microinjection plus insemination compared with subzonal sperm microinjection alone (Table 3). The higher fertilization rates failed to be maintained in the 24 patients who had subzonal sperm microinjection plus insemination only (Table 3). When comparing the subzonal sperm micro injection and subzonal sperm microinjection plus insemination techniques in patients who had failed % o (196) (51) No. of sperm injected Fi~~e ~ The effect of number of sperm microinjected into the perlvltelhne space on monospermic (_) and polyspermic (Il'i!) fertilization in the Melbourne trial. (Number of eggs injected are in parentheses above bar). Vol. 57, No.6, June 1992 Sakkas et al. Subzonal sperm microinjection 1283

6 Table 3 Fertilization Results of Patients Treated by Subzonal Sperm Microinjection Only, Subzonal Sperm Microinjection and Sub zonal Sperm Microinjection Plus Insemination, or Subzonal Sperm Microinjection Plus Insemination Only in the Melbourne and Bologna Trials Melbourne Technique used No. of No. of for patients cycles eggs Fertilized I-SUSM* only (loalt II-SUSM and (13.2) SUSMplus 35 9S 2S (2S.6) insemination III -SUSM plus IS (13.6) insemination only * Subzonal sperm micro injection. t Values in parentheses are percents. Bologna No. of No. of 2PN cycles eggs Fertilized 2 PN 25 (SA) (23.S) 24 (22.0) 11 {10.4):I: 72 6 (S.3) 6 (S.3) 22 (22A):j: 12 7S 9 (11.5) 9 (11.5) 16 (12.1) (S.O) 4 (S.O) :j: X 2 = 4.62; P < previous cycles, 3 of 30 (10.0%) and 5 of 23 (21.7%) 2 PN eggs were obtained, respectively. In patients selected because of poor semen quality, 8 of 76 (10.5%) and 17 of 75 (22.7%) 2 PN eggs were observed after subzonal sperm micro injection and sub zonal sperm micro injection plus insemination, respectively. Because the possibility existed that the surrounding sperm may be entering through the hole created by the microinjection pipette, 13 ofthe patients' eggs were allocated to three groups: (1) subzonal sperm micro injection only; (2) sub zonal sperm microinjection plus insemination; and (3) subzonal sperm microinjection without injection of sperm into the perivitelline space plus insemination, which resulted in fertilization rates of2 of29 (6.9%),8 of37 (21.6%), and 2 of 25 (8.0%) for the respective groups. In the Bologna trial, no significant difference was observed in the 12 patients receiving both subzonal sperm micro injection and subzonal sperm microinjection plus insemination (Table 3). This, however, may have been because of the use of Petri dishes, which had an increased volume compared with the wells used in the Melbourne trial; hence, the sperm suspension may have been overdiluted. Semen Quality and Fertilization Rate In this analysis, all the eggs treated by subzonal sperm microinjection alone from patient groups I and II were assessed for the Melbourne trial. For the Bologna trial, all eggs were pooled because there was no difference between subzonal sperm microinjection and subzonal sperm microinjection plus insemination. Normal Semen Compared With Male Factor Semen In the Melbourne trial, a number of excess eggs were donated by patients undergoing routine IVF of which 9 of 43 (20.9%) fertilized, including one polyspermic, after treatment by sub zonal sperm microinjection. A total of 403 eggs were treated by subzonal sperm micro injection from patient groups I and II of which 36 (8.9%) had 2 PN, significantly less (X 2 = 4.9; P < 0.05) than the number of 2 PN achieved when subzonal sperm microinjection was performed with patients who had normal semen parameters. Sperm Count There was no significant difference when comparing the fertilization rate and number of 2 PN after subzonal sperm microinjection with the initial count of sperm in the patient's ejaculate. In the Melbourne trial, 39 cycles were performed on patients with <5 X 10 6 sperm/ml resulting in 18 (9.0%) 2 PN of201 oocytes injected. Twenty-six cycles were performed on patients with between 5 and 20 X 10 6 sperm/ml, resulting in 14 (11.2%) 2 PN of 125 oocytes injected. In the Bologna trial, 21 «5 X 10 6 sperm/ml) and 16 (5 to 20 X 10 6 sperm/ml) cycles were performed, resulting in 24 of 186 (12.9%) and 14 of 98 (14.3%) 2 PN, respectively. Sperm Motility In the Melbourne trial, of all the eggs treated by subzonal sperm microinjection when the male had <50% motile sperm, 20 of 245 (8.1 %) were 2 PN eggs, whereas patients with 50% or more motile sperm had 13 of 144 (9.0%) 2 PN eggs (Fig. 2). In the Bologna trial, 30 patients had <50% motile 1284 Sakkas et al. Subzonal sperm microinjection Fertility and Sterility

7 % 2PN o < (67) (39) % motility Figure 2 The influence of percentage motility on the monospermic fertilization rate (2 PN) after eggs were treated by subzonal sperm microinjection (_) and subzonal sperm microinjection plus insemination (11!!1). Parentheses above bar contain the number of eggs treated and parentheses below bar contains total number of patients. (*P < 0.05). sperm, resulting in 32 of 253 (12.6%) 2 PN eggs after subzonal sperm microinjection, whereas 11 patients had 50% or more motile sperm with 11 of 56 (19.6%) 2 PN eggs. This trial also included the successful fertilization of a patient with immotile sperm and that of a patient after sperm were recovered from the epididymis. The motility of the patient's sperm in the initial ejaculate had no significant effect on fertilization rate after subzonal sperm microinjection alone. However, in the Melbourne trial, when the fertilization rates of all eggs treated by subzonal sperm micro injection alone and subzonal sperm microinjection plus insemination are compared, a significant increase in the percentage of monospermic fertilization is achieved in patients who had an initial motility of >50% (Fig. 2). (62) in correlating fertilization rates with the percentage of abnormal forms in the semen (Fig. 3). Sperm Antibodies Ten patients had varying degrees of sperm antibodies of which 5 achieved fertilization. There was no difference in the ability of sperm with antibodies to fertilize because 8 of 52 (15.4%) of the eggs injected fertilized. Unexplained Infertility In addition to anomalies in their semen parameters' 17 of the patients included in the Melbourne trial had unexplained infertility and had previously attempted three or more IVF cycles, failing to achieve fertilization. Of these patients, 9 (52.9%) achieved fertilization with a total of12 of 98 (12.2%) of the eggs fertilizing, of which 8 were 2 PN. Subzonal Sperm Microinjection Compared WithIVF Included in the Bologna trial were eight patients who had eggs allocated to both subzonal sperm microinjection and IVF. Of these patients' eggs, 12 of 43 (27.9%) fertilized after microinjection, whereas only 2 of 46 (4.3%) fertilized after routine IVF (x 2 = 7.6; P < 0.01). DISCUSSION The initial investigation conducted in this trial examined whether injection of multiple numbers of % 2PN 30 (58) Sperm Morphology In the Bologna trial, 23 patients had <50% abnormal sperm, resulting in 21 of 167 (12.6%) 2 PN eggs, whereas 10 patients had 50% or more abnormal sperm with 12 of 76 (15.8%) 2 PN eggs. This was also observed in the Melbourne trial in which the percentage of abnormal sperm did not affect the number of 2 PN obtained after subzonal sperm microinjection alone (Fig. 3). However, in the Melbourne trial, when percentage of abnormal forms are examined in connection with the fertilization rates of all eggs treated by subzonal sperm microinjection compared with all eggs treated by subzonal sperm microinjection plus insemination from patient groups I, II, and III, a significant difference is evident o < 50 (31) % abnormals 2 50 (60) Figure 3 The influence of the percentage of abnormal sperm On the monospermic fertilization rate (2 PN) after eggs were treated by subzonal sperm microinjection (_) and subzonal sperm microinjection plus insemination (11!!1). Parentheses above bar contain the number of eggs treated and parentheses below bar contain total number of patients. (*P < 0.05; **P < 0.05). Vol. 57, No.6, June 1992 Sakkas et ai. Subzonal sperm microinjection 1285

8 sperm under the zona could increase fertilization rates. A previous study had shown that there was an increase in polyspermy after injection of 10 to 12 human sperm under the zona of hamster and human eggs (16), whereas Trounson et al. (17) reported the same phenomena in the fertilization rate after injection of 8 to 11 mouse sperm into the perivitelline space of mouse oocytes. In addition, Cohen et al. (18) found that 6 of 9 fertilized eggs were polyspermic after microinjection into the perivitelline space with between 3 and 5 sperm and that all fertilized eggs were polyspermic when 9 or more sperm were injected. In this study, no real benefit was observed in the rate of monospermic fertilization after injection of up to 10 sperm under the zona. In agreement with other studies, there was a tendency to an increase in polyspermy when multiple numbers of sperm were injected. The risk of polyspermy is also prevalent in other zona invasive techniques that are used to treat male factor infertility (5, 9). Gordon et al. (5) reported that 10 of 47 (21 %) eggs fertilized after zona drilling; however, 5 of 10 displayed polyspermy. Unlike the techniques in which treatment relies on disrupting the zona so that sperm can easily enter the perivitelline space, the amount of sperm can be strictly controlled during the micro injection procedure, limiting the risk of polyspermy. In this study, it appears that the injection of 4 sperm was optimal in increasing fertilization while not jeopardizing the risk of polyspermy. As a modification to subzonal sperm microinjection alone, we also adopted a technique using subzonal sperm microinjection with the incubation of the injected oocyte with the remainder of the sperm suspension. The hole created by the microinjection procedure, unlike that in partial zona disection or zona drilling, was not large enough to increase the risk of polyspermy. Indeed, the low fertilization results obtained when sub zonal sperm microinjection was performed without injection of sperm and the eggs incubated in a sperm suspension did not allow us to make a conclusion on whether the sperm passed through the hole or were fertilized by sperm passing through the zona. In addition to facilitating the passage of sperm through the zona, it has been suggested that the hole in the zona may in fact be beneficial to the embryos' ability to implant (9), although the low implantation rate achieved in this study seems to contradict this suggestion. The question of whether the hole in the zona is deleterious to the embryos' chances of implanting, because of leucocytic invasion, is still unanswered. In this study, the overall fertilization rate remained <20%; hence, the majority of patients had only one or two embryos transfered, limiting the possibility of pregnancy. A total of 102 embryos were transfered, resulting in only three fetal heart beats (2.9%), one of which led to an abortion. The low number of pregnancies and three biochemical pregnancies after subzonal sperm micro injection warrant speculation on whether oocytes fertilized by male factor sperm are less viable than oocytes fertilized after routine IVF. Fishel et al. (19) reported that two of six pregnancies aborted after transfer of embryos obtained after microinjection. However, studies conducted in our Melbourne group have shown that pregnancies resulting after IVF with triple defect male factor patients did not exhibit an increase in abortion rate. In contrast to sub zonal sperm microinjection techniques in which the sperm are selected by an operator, IVF with male factor patients and partial zona disection allow a more vigorous sperm selection to occur whereby the fittest sperm should fertilize the egg. Therefore, when using poor semen in IVF and partial zona disection, embryo viability may not be affected, whereas in subzonal sperm micro injection and similar techniques, selection of poor quality sperm that fertilize the egg may in turn limit embryo viability. Indeed, it appears that quite a range of sperm are capable of achieving fertilization because in this study fertilization was achieved with patients containing antibodies and after injection of immotile sperm. Although the majority of patients treated had double or triple defects in semen quality, examination of the individual semen characteristics and their respective fertilization rates indicated that irrespective of the initial sperm quality, the chance of fertilization was the same for patients when treated by subzonal sperm microinjection. In contrast, comparison of subzonal sperm micro injection in male factor patients with patients with normal semen parameters showed that a higher fertilization rate was achieved using semen of normal quality. A correlation between fertilization after sub zonal sperm microinjection and semen quality may not be observed because when selecting a sperm for sub zonal sperm microinjection, the operator attempts to select sperm of normal morphology and motility. The techniques used to harvest a motile sperm population also would tend to harvest sperm of similar characteristics. This may result in the operator selecting a similar type of sperm in the majority of patients. In subzonal sperm microinjection, motility may not be necessary because fertilization has been reported previously using sperm from a patient with 1286 Sakkas et al. Subzonal sperm micro injection Fertility and Sterility

9 Kartagener's syndrome (20) and in this study after injection of immotile sperm. In contrast, unlike when subzonal sperm microinjection alone was used, patients in the Melbourne trial with >50% motility and <50% abnormal forms tended to have a higher chance of achieving fertilization when their eggs were placed in a dilute insemination droplet. The observation that subzonal sperm microinjection plus insemination was more successful in cases in which the sperm parameters were more advantageous may be because of two reasons. First, the sperm may have simply passed through the hole as is the case in the partial zona disection technique; this is supported by the fact that a small percentage of eggs were fertilized after being placed in dilute insemination after having a hole placed in the zona. Second, the observation that poor sperm quality is not advantageous when subzonal sperm microinjection plus insemination is performed may be because of defective sperm populations being present in the sperm suspension. Studies by Aitken and Clarkson (21, 22) have shown the existence of populations of sperm in an ejaculate that produce superoxide and hydroxyl radicals that induce irreversible damage to spermatozoa and impair their fertilizing ability. This phenomena and its effect on fertilization when treating males with poor semen parameters by micromanipulation techniques is currently under investigation. In agreement with this observation, Cohen et al. (9) also reported that less severe male factor patients with only single defects, oligozoospermia, or asthenozoospermia had a higher fertilization rate when compared with patients with oligoasthenozoospermia when treated using the partial zona disection technique. The technical variations investigated in this study failed to provide a dramatic increase in fertilization rates. Therefore, the preparatory methods of semen before micro injection must be improved so that the chances of selecting a sperm capable of fertilizing are maximized. A major hurdle of the sub zonal sperm micro injection procedure is that it eliminates exposure of the sperm to both the cumulus cells and the zona pellucida. Both the zona pellucida and the cumulus cells have been reported to influence the acrosomal status of human sperm before entering the egg (23, 24). Cross et al. (23) reported that the rate of acrosome reactions increased from 3% in control to 24% when sperm were treated in solubilized human zonae. In addition, a 50,000 molecular weight fraction of cumulus cell-conditioned medium has also been shown to stimulate the acrosome reaction of human sperm (25). Clearly, for microin- jection to be more successful, a greater percentage of the sperm must be prepared in such a way to increase the chances of fertilization when they are placed into the perivitelline space of the oocytes. This may be achieved by techniques such as incubation of sperm with zona pellucida proteins or cumulus cells before injection to overcome the lack of exposure to the oocyte vestments. The zona drilling technique and partial zona disection technique also eliminate exposure of sperm to the zona or cumulus cells; however, in these techniques a greater number of sperm pass into the perivitelline space, readily increasing the probability that an acrosome-reacted sperm capable of fertilization is in the vicinity of the oolema. The subzonal sperm microinjection technique, along with other micromanipulation techniques, have been successfully adopted to improve the chance of pregnancy in couples in whom the male has severely reduced semen quality. This trial has resulted in pregnancies after micro injection of male factor sperm and provided valuable data for the investigation of the sub zonal sperm micro injection technique. The technical modifications to the microinjection technique investigated in this study, however, failed to provide any dramatic increase in the fertilization rate of injected oocytes; hence, the number of embryos transferred remains low, limiting the chance of a pregnancy. Therefore, for the subzonal sperm microinjection technique, further improvements should now be aimed at the preparation techniques used before the injection of sperm so that the majority of sperm are primed to fertilize the egg, limiting the probability of selecting sperm that are not ready or capable of fertilization. Acknowledgments. We gratefully acknowledge the assistance of the staff at the Infertility Medical Centre, Epworth, Melbourne, Australia, and the IVF Centre, Villa Regina, Bologna, Italy. REFERENCES 1. 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