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Fertilization after standard in vitro fertilization versus intracytoplasmic sperm injection in subfertile males using sibling oocytes
FERTILITY AND STERILITYt VOL. 71, NO. 4, APRIL 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Fertilization after standard in vitro fertilization versus intracytoplasmic sperm injection in subfertile males using sibling oocytes Margareta D. Pisarska, M.D.,* Peter R. Casson, M.D.,* Pauline L. Cisneros, Ph.D.,* Dolores J. Lamb, Ph.D.,†‡ Larry I. Lipshultz, M.D.,† John E. Buster, M.D.,* and Sandra A. Carson, M.D.* Baylor Assisted Reproductive Technology Program, Baylor College of Medicine, Houston, Texas
Objective: To compare conventional IVF with ICSI in the subfertile male population using sibling oocytes. Results from males with isolated severe teratozoospermia also are analyzed. Design: Prospective experimental study. Setting: University based IVF clinic. Patient(s): Group A: 18 patients with one or more abnormalities in count, motility, or morphology. Group B: 20 patients with isolated severe teratozoospermia (#4% Kruger Strict Criteria). Intervention(s): Ovulation induction, random allocation of sibling oocytes, and IVF or ICSI. Main Outcome Measure(s): Fertilization rates (fertilization per cycle, fertilization per oocytes, and fertilization per couple) and embryo quality.
Received July 16, 1998; revised and accepted November 23, 1998. Presented at the 53rd Annual Meeting of the American Society of Reproductive Medicine, Cincinnati, Ohio, October 18 –22, 1997. Reprint requests: Sandra A. Carson, M.D., Department of Obstetrics and Gynecology, Baylor College of Medicine, 6550 Fannin, Suite 801 Houston, Texas 77030 (FAX: 713798-8431; E-mail: [email protected]). * Department of Obstetrics and Gynecology. † Scott Department of Urology. ‡ Department of Cell Biology. 0015-0282/99/$20.00 PII S0015-0282(98)00538-X
Result(s): In group A, fertilization occurred in 13 of 18 (72%) of IVF cycles and 17 of 18 (94%) of ICSI cycles. Overall, 69 of 120 (58%) oocytes fertilized after IVF, whereas 80 of 131 (61%) fertilized after ICSI. The mean (6SEM) percent of oocytes fertilized per couple was 44.6% 6 9.0% with IVF and 62.7% 6 5.6% with ICSI (not statistically significant). In group B, fertilization occurred in 18 of 20 (90%) cycles after IVF and 20 of 20 (100%) cycles with ICSI. Overall, 54 of 113 (48%) of the oocytes fertilized after IVF, whereas 82 of 124 (66%) fertilized with ICSI. The mean (6SEM) percent of oocytes fertilized per couple was 50.9% 6 7.1% with IVF and 66.6% 6 4.7% with ICSI. No statistically significant difference in embryo quality after IVF versus ICSI was demonstrated. Conclusion(s): With severe teratozoospermia, ICSI results in higher fertilization rates than conventional IVF, without altering embryo quality. In our subfertile male population, there is a trend toward improved fertilization with ICSI, with less failed fertilization. (Fertil Sterilt 1999;71:627–32. ©1999 by American Society for Reproductive Medicine.) Key Words: Subfertile males, teratozoospermia, sibling oocytes, in vitro fertilization, intracytoplasmic sperm injection, fertilization, embryo quality
Intracytoplasmic sperm injection (ICSI) with its high fertilization and pregnancy rates has replaced conventional IVF and other types of micromanipulation as first-line therapy in couples with severe male factor infertility (1– 4). The high success rate of ICSI has led to extension of the technique to other patient populations for whom conventional IVF may be an option, including subfertile males and unexplained infertility (5). Furthermore, ICSI has been tried solely to improve embryo quality in couples without male factor infertility, without
considering costs and the need for qualified laboratory personnel and facilities (6). One group of infertile couples for whom the use of ICSI may be superior to conventional IVF is when severe teratozoospermia is the only semen abnormality because severe teratozoospermia has been implicated as the most important factor affecting fertilization with conventional IVF (7, 8). Therefore, we evaluated IVF versus ICSI with use of sibling oocytes from partners of subfertile males to assess fertilization and embryo quality. Further 627
analysis was performed on the specific group of males with severe teratozoospermia in the presence of normal count and motility.
MATERIALS AND METHODS Patient Selection Subfertile males were defined by the presence of at least one abnormal semen parameter, i.e., count of ,20 3 106/mL or motility of ,40% according to the World Health Organization (WHO) criteria, regardless of morphology according to Kruger’s strict criteria (7, 9). In this population, the mean (6SEM) count was 22.3 6 9.4 3 106 sperm/mL, with a range of 1–33 3 106 sperm/mL. One patient had a count of 179 3 106 sperm/mL. This patient was included because of low motility. Mean (6SEM) motility was 45.0% 6 3.5%, with a range of 25%–75%, and mean (6SEM) motile sperm concentration was 8.0 6 2.3 3 106 motile sperm/mL. All patients had a count of $1 3 106/mL and motility of $25%. A total of 18 couples were selected to undergo an IVF cycle in which oocytes were divided without prejudice between conventional IVF and ICSI. Severe teratozoospermia (morphology of #4% normal forms according to Kruger’s strict criteria [7, 9]) in the presence of normal count and motility (WHO criteria) was analyzed separately. Couples (n 5 20) in whom severe teratozoospermia was the only semen abnormality also were selected to undergo an IVF cycle in which oocytes were divided without prejudice between conventional IVF and ICSI.
Ovulation Induction All patients underwent pituitary down-regulation with SC 1 mg/d of leuprolide acetate (Lupron; TAP Pharmaceuticals Inc., Deerfield, IL) starting on day 21 of the cycle before follicular stimulation with menotropins (Pergonal; Serono Laboratories, Randolph, MA). At the time of menotropin administration, the SC leuprolide acetate dosage was decreased to 0.5 mg/d. Stimulation was monitored with transvaginal ultrasound and E2 levels. After follicle maturity was ascertained (leading follicle diameter of $20 mm; E2 level of .200 pg/mL per mature follicle), 10,000 IU of hCG (Profasi; Serono Laboratories) was administered intramuscularly. After 34 –36 hours, oocyte aspiration was performed under transvaginal ultrasound guidance with use of intravenous sedation.
Spermatozoa Preparation On the day of aspiration, semen samples were assessed for concentration (sperm/mL) and motility (% motile). The sample was prepared on two-layer Percoll gradient (Sigma, St. Louis, MO). The count and motility of the postwash sample were adjusted to 0.15 3 106 sperm/mL in human tubal fluid (HTF; Irvine Scientific, Irvine, CA) and 8% maternal serum for IVF. Higher concentrations, 0.3 3 106 628
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sperm/mL, were used in some cases when semen parameters were poor. The tube was capped loosely and set in the incubator at 37°C, 5% CO2 in air until the time of insemination. The sample for ICSI was prepared by the addition of 0.3 mL of modified sperm wash media (MSWM; Irvine Scientific) to the pellet. The tube was tightly capped and kept at 37°C until the time of sperm injection.
Oocyte Preparation and Insemination Oocytes were assigned randomly to either IVF or ICSI. The IVF oocytes were kept in 100-mL drops of HTF and 8% maternal serum. Five to six hours postaspiration, one oocyte was placed in 15,000 sperm per 100-mL drop. The ICSI oocytes were placed in hyaluronidase to remove the cumulus, rinsed in HTF and maternal serum and stripped of the corona cells. Only oocytes with a polar body were injected. Several injection dishes were prepared with one drop of polyvinyl pyrrolidone (Irvine Scientific) and one drop of MSWM. Each oocyte was injected with sperm of normal morphology displaying motility on Nikon-Narishige micromanipulation equipment (Melville, NY) using injection and holding pipettes from Humagen Fertility Diagnostics (Charlottesville, VA). Once injection was completed, the oocytes were rinsed in HTF and maternal serum.
Fertilization and Embryo Quality On day 1 (16 –20 hours after retrieval) the oocytes were checked for fertilization. The IVF oocytes were stripped to remove the corona. The presence of more than 2 pronuclei (2PN) was noted. Oocytes with 2PN were transferred to growth media (HTF 1 15% maternal serum). On day 2 (40 – 43 hours after retrieval), embryo quality was recorded by cell number and cell grade. Cell grade was classified according to the following modified D. P. Wolf criteria (10). Grade 4 and 5 embryos are considered superior quality embryos, with the blastomeres evenly shaped and equal in size. Grade 5 embryos contain no cytoplasmic fragments, and grade 4 embryos have cytoplasmic fragments consisting of ,10% of the embryo volume. Grade 3 embryos have blastomeres with slightly distorted shapes and sizes that are slightly uneven. Cytoplasmic fragments may be present in up to 25% of the embryo volume. Grade 2 embryos contain significantly distorted blastomeres that are unequal in size, and the cytoplasmic fragments make up 25%–50% of the embryo volume. The poorest quality embryos, grade 1, have uneven fragmented blastomeres with .50% of the embryo volume consisting of cytoplasmic fragments. The ooplasm is dark and flat, distinct from the nongranular and nonstatic ooplasm of the other grades. For the purposes of this study, a scoring system was developed incorporating cell number and cell grade (Table 1).
Embryo Transfer After oocyte aspiration, prednisone (10 mg b.i.d.) and tetracycline (250 mg q.i.d.) was administered for 4 days. Vol. 71, No. 4, April 1999
TABLE 1
TABLE 2
Embryo quality scoring system. Cell no.
Comparison of IVF and ICSI from sibling oocytes in subfertile males.
Cell grade*
Score
5 4 5 4 5 4 5 4 5 4 3 3 3 3 3 2 2 2 2 2 1
1 Best quality 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Poorest quality
Treatment 6 6 5 5 4 4 3 3 2 2 6 5 4 3 2 6 5 4 3 2 Any cell number 2PN Note: PN 5 pronuclei. * Modified D. P. Wolf criteria.
Progesterone in oil 25 mg (50 mg/mL; Steris Laboratories, Phoenix, AZ) was given for daily luteal support starting the day of ET and continued until menses or 8 gestational weeks. Up to six embryos were transferred with use of a Wallace catheter.
Pregnancy Assessment
Serum b-hCG was obtained 14 days after aspiration, and confirmatory ultrasound was performed 2 weeks later. Clinical pregnancy was defined as the presence of a sac at 5 weeks of gestation.
Statistical Analysis Fisher’s exact test was used to compare the number of successful cycles with IVF versus ICSI. The oocyte fertilization rate was calculated as the number of fertilized oocytes divided by the number of metaphase II oocytes inseminated. The overall number of oocytes fertilized with IVF versus ICSI was compared with use of the x2 test, and percentage of oocytes fertilized per couple were compared between IVF and ICSI by the Wilcoxon matched-pairs test. Embryo quality, based on cell number, grade, and score was compared with the Mann-Whitney U-test, and the x2 test was used to compare superior quality embryos (based on grade and score) between IVF and ICSI. This protocol was approved by the Institutional Review Board at Baylor College of Medicine and affiliated hospitals. FERTILITY & STERILITYt
Variable No. of cycles with fertilization/total no. of cycles (%) No. of oocytes fertilized/no. of oocytes allocated (%) Mean (6SEM) percentage of oocytes fertilized No. of grade 4 or 5 embryos/total no. of embryos (%) * † ‡ §
P P P P
5 5 5 5
not not not not
significant significant significant significant
(determined (determined (determined (determined
by by by by
IVF
ICSI
13/18 (72)*
17/18 (94)*
69/120 (58)†
80/131 (61)†
44.6 6 9.0‡
62.7 6 5.6‡
32/69 (46)§
38/74 (51)§
Fisher’s exact test). x2-test). Wilcoxon matched-pairs test). x2-test).
RESULTS Subfertile Male Group (Group A) A total of 18 couples with one or more abnormal semen parameters underwent an IVF cycle in which oocytes were divided between conventional IVF and ICSI (Table 2). Fertilization occurred in 13 of 18 (72%) of cycles with IVF and 17 of 18 (94%) of cycles with ICSI (P5.18). The single cycle with failed fertilization after ICSI also had failed fertilization with conventional IVF. A total of 120 metaphase II oocytes were allocated to IVF, and 131 metaphase II oocytes were allocated to ICSI. Overall, 69 of 120 (58%) of the oocytes fertilized with IVF, whereas 80 of 131 (61%) fertilized with ICSI (P 5 .57). Of the total cohort injected, the damage rate was 17 of 131 (13%). The mean (6SEM) percentage of oocytes fertilized per couple was 44.6% 6 9.0% with IVF and 62.7% 6 5.6% with ICSI (P 5 .09). Of the 18 couples, 7 had better fertilization with IVF than ICSI. Comparisons between percent fertilization and semen parameters were made. There was no correlation between fertilization rates per couple with either IVF or ICSI and sperm count, motility, or motile sperm concentration. There was no statistically significant difference in embryo quality based on cell number, cell grade, and score between embryos from IVF versus ICSI. Thirty-two of the 69 (46%) embryos from IVF were either grade 4 or 5 (equivalent to a score of #10). Similarly, 38 of 74 (51%) of embryos from ICSI were grade 4 or 5. This was also not statistically significant. Differences in pregnancy rates with IVF versus ICSI could not be assessed because embryos were transferred concurrently from both procedures. Eleven of the 18 (61%) couples achieved pregnancy. Currently, there are four ongoing intrauterine pregnancies, seven delivered intrauterine 629
DISCUSSION
TABLE 3 Comparison of IVF and ICSI from sibling oocytes in males with severe teratozoospermia. Treatment Variable No. of cycles with fertilization/total no. of cycles (%) No. of oocytes fertilized/no. of oocytes allocated (%) Mean (6SEM) percentage of oocytes fertilized No. of grade 4 or 5 embryos/total no. of embryos (%) * † ‡ §
IVF
ICSI
18/20 (90)*
20/20 (100)*
54/113 (48)†
82/124 (66)†
50.9 6 7.1‡
66.6 6 4.7‡
16/54 (30)§
24/79 (30)§
P 5 not significant (determined by Fisher’s exact test). P5.004 (determined by x2-test). P5.04 (determined by Wilcoxon matched-pairs test). P 5 not significant (determined by x2-test).
pregnancies, and one second trimester loss secondary to chorioamnionitis. There were seven multiple gestations. Two intrauterine pregnancies (1 set of term twins and the second trimester twin loss) were achieved in couples who had fertilization with ICSI only.
Teratozoospermia Group (Group B) Twenty couples for whom severe teratozoospermia (morphology of #4% normal forms) was the only identifiable cause of subfertility were separately analyzed (Table 3). Fertilization occurred in 18 of 20 (90%) cycles with IVF and 20 of 20 (100%) cycles with ICSI (P5.49). A total of 113 metaphase II oocytes were allocated to IVF, and 124 metaphase II oocytes were allocated to ICSI. Overall, 54 of 113 (48%) of the oocytes fertilized with IVF, whereas 82 of 124 (66%) fertilized with ICSI (P5.004). Of the total cohort injected, the damage rate was 17 of 124 (14%). The mean (6SEM) percentage of oocytes fertilized per couple was 50.9% 6 7.1% with IVF and 66.6% 6 4.7% with ICSI (P5.04). There was a statistically significant difference in the overall number of oocytes fertilized with IVF versus ICSI and the percentage of oocytes fertilized per couple. There was no statistically significant difference in embryo quality based on cell number, cell grade, and score between embryos from IVF versus ICSI. Sixteen of the 54 (30%) embryos from IVF were either grade 4 or 5 (equivalent to a score of #10), and 24 of 79 (30%) of embryos from ICSI were grade 4 or 5. Seven of the 20 (35%) couples achieved pregnancy. Currently, there are five ongoing singleton pregnancies, one term infant, and one ectopic pregnancy—an intrauterine pregnancy rate of 30%. One ongoing pregnancy was achieved in a couple who had fertilization with ICSI only. 630
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In men whose only semen abnormality was severe teratozoospermia, ICSI resulted in a statistically superior overall number of oocytes fertilized and percentage of oocytes fertilized per couple, compared with conventional IVF, without altering embryo quality. However, ICSI did not significantly improve fertilization or embryo quality compared with conventional IVF of sibling oocytes in this population of subfertile males, defined by the presence of at least one abnormal semen parameter (count of ,20 3 106/mL or motility of ,40% [WHO]) regardless of morphology (Kruger’s strict criteria [7, 9]). By using sibling oocytes, we were able to remove any female factor that might affect fertility, resulting in a more accurate evaluation of IVF versus ICSI in this infertile population. The number of oocytes fertilized per couple between IVF and ICSI constitutes the strength of this study and was not performed in previous sibling oocyte comparisons. There was no significant difference after ICSI in the number of cycles with fertilization, overall number of oocytes fertilized, and percent oocytes fertilized per couple, in this population of subfertile males. However, only 1 couple of 18 had failed fertilization with ICSI, whereas 5 of 18 couples had failed fertilization with IVF. Further evaluation of this population did not reveal any predictors of poor outcome. We did not demonstrate the same statistically increased overall number of oocytes fertilized with ICSI compared with conventional IVF in a subfertile male population as did Aboulghar et al. (5). In the former study, also using sibling oocytes, subfertile males were defined as having at least one normal semen analysis based on WHO criteria and at least one subnormal parameter on another semen analysis. In this group of 24 couples, the overall number of oocytes fertilized with conventional IVF was 27.1% and 59% with ICSI. However, these investigators did not pair the data to statistically analyze fertilization in each couple, the strength of using sibling oocytes. The most important semen parameter that affected fertilization when comparing conventional IVF with ICSI was morphology in the presence of normal count and motility. In couples with severe teratozoospermia, #4% normal forms by Kruger’s strict criteria, ICSI increased the number of oocytes fertilized by 18% (P5.004) compared with IVF, and the fertilization rate per couple was increased from 50.9% to 66.6% (P5.04). It is surprising that only two couples had complete fertilization failure with conventional IVF, and none of the couples had failed fertilization with ICSI. This is consistent with the widely held tenet that morphology is one of the most important semen parameters predicting fertilization in conventional IVF. Initially, the overall number of oocytes fertilized was as low as 7.6% in Vol. 71, No. 4, April 1999
patients with severe teratozoospermia (7). Improvements in IVF has led to higher numbers of oocytes fertilized in couples with severe teratozoospermia, but these rates still remain significantly lower than in couples with a strict morphology of .4% (8, 11). Ombelet et al. (11) assessed IVF success based on sperm morphology. They found that the overall number of oocytes fertilized in couples with ,9% normal forms, based on Kruger’s strict criteria, was 69.2% compared with 79.4% in couples with .9% normal forms. Ombelet et al. (11) further divided these patients with ,9% normal forms into a poor prognosis group with ,5% normal forms based on a fertilization rate per oocyte of 37.9% with a 0% pregnancy rate and a good prognosis group, 5%– 8% normal forms, with a fertilization rate per oocyte of 78.6% and a 15.8% pregnancy rate. However, this group did not use sibling oocytes. Unlike IVF, ICSI is not dependent on any semen parameters (3, 12) including morphology. Mansour et al. (13) found no significant difference in fertilization and pregnancy rates between patients with ,5% normal forms using strict criteria with a 59% fertilization rate per oocyte and 36.6% pregnancy rate and patients with .5% normal forms with a 57.3% fertilization rate per oocyte and a pregnancy rate of 34.8%. Again, no sibling oocytes were used. Although teratozoospermia has been associated with poor fertilization using conventional IVF, whereas fertilization with ICSI is not affected routinely, none of these studies used sibling oocytes to account for uncontrollable differences between groups. Only one other study evaluated “conventional” IVF with ICSI in sibling oocytes in men with poor morphology (2). Unlike our study, poor morphology was defined as ,20% normal forms based on WHO criteria. Furthermore, poor semen morphology was not always the only abnormal semen parameter. This may account for the study’s extremely poor overall fertilization rate per oocyte of 15%, with only 39% of cycles with fertilization, using conventional IVF compared with an overall fertilization rate per oocyte of 76%, with 100% of cycles with fertilization, when ICSI was used. Similar findings were observed in studies, using sibling oocytes, when conventional IVF was replaced with high insemination concentration IVF. Fishel et al. (14) found that 37% of oocytes fertilized with high insemination concentration IVF versus 58% with ICSI in patients with ,5% normal forms. The number of oocytes fertilized in patients with .5% normal forms were equivalent whether high insemination concentration IVF or ICSI was used. However, Fishel’s patient population failed prior conventional IVF, either on two previous occasions or on a single occasion in which there was no binding to the zona pellucida. This group was even more selective than the group in our study and may represent the subpopulations in our group that benefit using ICSI. Nonetheless, it would be impractical to FERTILITY & STERILITYt
let patients fail one IVF cycle before subjecting them to IVF and ICSI. Only one study found superior fertilization rates per oocyte with high insemination concentration IVF over ICSI, 86% versus 68%, respectively. Unfortunately, this study did not evaluate sibling oocytes (15). Again, even in those studies mentioned above, no paired statistical analysis was performed to assess fertilization within each couple. We could not compare pregnancy rates as a result of IVF versus ICSI because couples in our study had embryos transferred from both procedures. However, we did not find any difference in embryo quality between IVF fertilized embryos and ICSI embryos in either group, the subfertile males or the teratozoospermic males. Embryo quality was based on a scoring system incorporating cell number and cell grade using modified D. P. Wolf criteria (10). One other study found similar results when oocytes were divided between high insemination concentration IVF and ICSI in couples with severe teratozoospermia (16). In contrast, Oehninger et al. (15) found that ICSI produced superior quality embryos, defined as grade 1 or 2, in couples with severe teratozoospermia, than with high insemination concentration IVF, 87% grade 1 and 2 embryos with ICSI compared with 72% with high insemination concentration IVF. Similarly, Fishel et al. (14) also found that in couples with severe teratozoospermia, high insemination concentration IVF resulted in compromised cleavage morphology over ICSI, only 30% of embryos after high insemination concentration IVF were normal compared with 84% with ICSI. However, these couples had a history of failed fertilization with conventional IVF and may represent a different subpopulation. Although counterintuitive, the embryo quality may be the result of the higher concentration of sperm decreasing embryo quality rather than ICSI improving it. In studies comparing IVF with ICSI in nonmale factor infertility, Yang et al. (6) found statistically more superior quality embryos, defined as grade A embryos, in the ICSI group than in the IVF group, 35.4% versus 24.3%, respectively. Yang et al. (6) explained the improved embryo quality because “ICSI technique avoids oocytes and zygotes cultured with a lot of spermatozoa (100,000 –500,000 spermatozoa per well), which does not happen in vivo. Human spermatozoa can produce reactive oxygen species such as superoxide anion (O22) and hydrogen peroxide (H2O2) (17, 18). It seems likely that free radical toxicity also contributes to embryonic damage during the culture period necessary for human IVF.” If free radical toxicity does contribute to embryonic damage, a smaller concentration of spermatozoa should be used, paradoxically, to improve embryo quality with IVF. Although there is a trend toward improved fertilization with ICSI in this specific population of subfertile males with low semen parameters, other than morphology, there is no 631
single parameter that may act as a predictor to determine outcome. Until such a predictor becomes available, based on our results, conventional IVF is an option in this population without the disadvantages of ICSI, including higher costs, increased time consumption, and personnel training. However, with increased cycles of failed fertilization with IVF compared with ICSI in this subpopulation, the desire to optimize chances of pregnancy may lead to ICSI as the preferred treatment option in this group of patients. One semen parameter that may act as a predictor for using ICSI as a first-line therapeutic modality is morphology. Severe teratozoospermia, defined as #4% normal forms according to Kruger’s strict criteria (7, 9), is the most significant factor that affects fertilization when IVF is used. Thus, ICSI should be used in this patient population with subsequent high fertilization rates and good embryo quality. References 1. Society for Assisted Reproductive Technology and the American Society of Reproductive Medicine. Assisted reproductive technology in the United States and Canada: 1995 results generated from the american society for reproductive medicine/society for assisted reproductive technology registry. Fertil Steril 1998;69:389 –98. 2. Payne D, Flaherty SP, Jeffrey R, Warnes GM, Matthews CD. Successful treatment of severe male factor infertility in 100 consecutive cycles using intracytoplasmic sperm injection. Hum Reprod 1994;9:2051–7. 3. Palermo G, Joris H, Derde MP, Camus M, Devroey P, Van Steirteghem AC. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil Steril 1993;59:826 –35. 4. Van Steirteghem AC, Liu J, Joris H, Nagy Z, Janssenswillen C, Tournaye H, et al. Higher success rate by intracytoplasmic sperm injection than by subzonal insemination: report of a second series of 300 consecutive treatment cycles. Hum Reprod 1993;8:1055– 60. 5. Aboulghar MA, Mansour RT, Serour GI, Sattar MA, Amin YM. Intracytoplasmic sperm injection and conventional in vitro fertilization for sibling oocytes in cases of unexplained infertility and borderline semen. J Assist Reprod Genet 1996;13:38 – 42.
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6. Yang D, Shahata MA, Al-Bader M, Al-Natsha SD, Al-Flamerzia M, Al-Shawaf T. Intracytoplasmic sperm injection improving embryo quality: comparison of sibling oocytes of non-male-factor couples. J Assist Reprod Genet 1996;13:351–5. 7. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1988;49:112–7. 8. Grow DR, Oehninger S, Seltman HJ, Toner JP, Swanson RJ, Kruger TF, et al. Sperm morphology as diagnosed by strict criteria: probing the impact of teratozoospermia on fertilization rate and pregnancy outcome in a larger in vitro fertilization population. Fertil Steril 1994;62:559 – 67. 9. Menkveld R, Stander FSH, Kotze TJ, Kruger TF, van Zyl JA. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 1990;5:586 –92. 10. Wolf DP, ed. In vitro fertilization and embryo transfer, a manual of basic techniques. New York: Plenum Press, 1988. 11. Ombelet W, Fourie FL, Vandeput H, Bosmans E, Cox A, Janssen M, et al. Teratozoospermia and in vitro fertilization: a randomized prospective study. Hum Reprod 1994;9:1479 – 84. 12. Cohen J, Talansky BE, Malter H, Alikani M, Adler A, Reing A, et al. Microsurgical fertilization and teratozoospermia. Hum Reprod 1991;6: 118 –23. 13. Mansour RT, Aboulghar MA, Serour GI, Amin YM, Ramzi AM. The effect of sperm parameters on the outcome of intracytoplasmic sperm injection. Fertil Steril 1995;64:982– 6. 14. Fishel S, Lisi F, Rinaldi L, Lisi R, Timson J, Green S, et al. Intracytoplasmic sperm injection (ICSI) versus high insemination concentration (HIC) for human conception in vitro. Reprod Fertil Dev 1995;7: 169 –75. 15. Oehninger S, Kruger TF, Simon T, Jones D, Mayer J, Lanzendorf S, et al. A comparative analysis of embryo implantation potential in patients with severe teratozoospermia undergoing in-vitro fertilization with a high insemination concentration or intracytoplasmic sperm injection. Hum Reprod 1996;11:1086 –9. 16. Hall J, Fishel S, Green S, Fleming S, Hunter A, Stoddart N, et al. Intracytoplasmic sperm injection versus high insemination concentration in-vitro fertilization in cases of very severe teratozoospermia. Hum Reprod 1995;2:493– 6. 17. Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa: superoxide dismutase as a major enzyme protectant against oxygen toxicity. J Androl 1987;8:338 – 48. 18. Atiken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 1987;81:459 – 69.
Vol. 71, No. 4, April 1999
Fertilization after standard in vitro fertilization versus intracytoplasmic sperm injection in subfertile males using sibling oocytes Margareta D. Pisarska, M.D.,* Peter R. Casson, M.D.,* Pauline L. Cisneros, Ph.D.,* Dolores J. Lamb, Ph.D.,†‡ Larry I. Lipshultz, M.D.,† John E. Buster, M.D.,* and Sandra A. Carson, M.D.* Baylor Assisted Reproductive Technology Program, Baylor College of Medicine, Houston, Texas
Objective: To compare conventional IVF with ICSI in the subfertile male population using sibling oocytes. Results from males with isolated severe teratozoospermia also are analyzed. Design: Prospective experimental study. Setting: University based IVF clinic. Patient(s): Group A: 18 patients with one or more abnormalities in count, motility, or morphology. Group B: 20 patients with isolated severe teratozoospermia (#4% Kruger Strict Criteria). Intervention(s): Ovulation induction, random allocation of sibling oocytes, and IVF or ICSI. Main Outcome Measure(s): Fertilization rates (fertilization per cycle, fertilization per oocytes, and fertilization per couple) and embryo quality.
Received July 16, 1998; revised and accepted November 23, 1998. Presented at the 53rd Annual Meeting of the American Society of Reproductive Medicine, Cincinnati, Ohio, October 18 –22, 1997. Reprint requests: Sandra A. Carson, M.D., Department of Obstetrics and Gynecology, Baylor College of Medicine, 6550 Fannin, Suite 801 Houston, Texas 77030 (FAX: 713798-8431; E-mail: [email protected]). * Department of Obstetrics and Gynecology. † Scott Department of Urology. ‡ Department of Cell Biology. 0015-0282/99/$20.00 PII S0015-0282(98)00538-X
Result(s): In group A, fertilization occurred in 13 of 18 (72%) of IVF cycles and 17 of 18 (94%) of ICSI cycles. Overall, 69 of 120 (58%) oocytes fertilized after IVF, whereas 80 of 131 (61%) fertilized after ICSI. The mean (6SEM) percent of oocytes fertilized per couple was 44.6% 6 9.0% with IVF and 62.7% 6 5.6% with ICSI (not statistically significant). In group B, fertilization occurred in 18 of 20 (90%) cycles after IVF and 20 of 20 (100%) cycles with ICSI. Overall, 54 of 113 (48%) of the oocytes fertilized after IVF, whereas 82 of 124 (66%) fertilized with ICSI. The mean (6SEM) percent of oocytes fertilized per couple was 50.9% 6 7.1% with IVF and 66.6% 6 4.7% with ICSI. No statistically significant difference in embryo quality after IVF versus ICSI was demonstrated. Conclusion(s): With severe teratozoospermia, ICSI results in higher fertilization rates than conventional IVF, without altering embryo quality. In our subfertile male population, there is a trend toward improved fertilization with ICSI, with less failed fertilization. (Fertil Sterilt 1999;71:627–32. ©1999 by American Society for Reproductive Medicine.) Key Words: Subfertile males, teratozoospermia, sibling oocytes, in vitro fertilization, intracytoplasmic sperm injection, fertilization, embryo quality
Intracytoplasmic sperm injection (ICSI) with its high fertilization and pregnancy rates has replaced conventional IVF and other types of micromanipulation as first-line therapy in couples with severe male factor infertility (1– 4). The high success rate of ICSI has led to extension of the technique to other patient populations for whom conventional IVF may be an option, including subfertile males and unexplained infertility (5). Furthermore, ICSI has been tried solely to improve embryo quality in couples without male factor infertility, without
considering costs and the need for qualified laboratory personnel and facilities (6). One group of infertile couples for whom the use of ICSI may be superior to conventional IVF is when severe teratozoospermia is the only semen abnormality because severe teratozoospermia has been implicated as the most important factor affecting fertilization with conventional IVF (7, 8). Therefore, we evaluated IVF versus ICSI with use of sibling oocytes from partners of subfertile males to assess fertilization and embryo quality. Further 627
analysis was performed on the specific group of males with severe teratozoospermia in the presence of normal count and motility.
MATERIALS AND METHODS Patient Selection Subfertile males were defined by the presence of at least one abnormal semen parameter, i.e., count of ,20 3 106/mL or motility of ,40% according to the World Health Organization (WHO) criteria, regardless of morphology according to Kruger’s strict criteria (7, 9). In this population, the mean (6SEM) count was 22.3 6 9.4 3 106 sperm/mL, with a range of 1–33 3 106 sperm/mL. One patient had a count of 179 3 106 sperm/mL. This patient was included because of low motility. Mean (6SEM) motility was 45.0% 6 3.5%, with a range of 25%–75%, and mean (6SEM) motile sperm concentration was 8.0 6 2.3 3 106 motile sperm/mL. All patients had a count of $1 3 106/mL and motility of $25%. A total of 18 couples were selected to undergo an IVF cycle in which oocytes were divided without prejudice between conventional IVF and ICSI. Severe teratozoospermia (morphology of #4% normal forms according to Kruger’s strict criteria [7, 9]) in the presence of normal count and motility (WHO criteria) was analyzed separately. Couples (n 5 20) in whom severe teratozoospermia was the only semen abnormality also were selected to undergo an IVF cycle in which oocytes were divided without prejudice between conventional IVF and ICSI.
Ovulation Induction All patients underwent pituitary down-regulation with SC 1 mg/d of leuprolide acetate (Lupron; TAP Pharmaceuticals Inc., Deerfield, IL) starting on day 21 of the cycle before follicular stimulation with menotropins (Pergonal; Serono Laboratories, Randolph, MA). At the time of menotropin administration, the SC leuprolide acetate dosage was decreased to 0.5 mg/d. Stimulation was monitored with transvaginal ultrasound and E2 levels. After follicle maturity was ascertained (leading follicle diameter of $20 mm; E2 level of .200 pg/mL per mature follicle), 10,000 IU of hCG (Profasi; Serono Laboratories) was administered intramuscularly. After 34 –36 hours, oocyte aspiration was performed under transvaginal ultrasound guidance with use of intravenous sedation.
Spermatozoa Preparation On the day of aspiration, semen samples were assessed for concentration (sperm/mL) and motility (% motile). The sample was prepared on two-layer Percoll gradient (Sigma, St. Louis, MO). The count and motility of the postwash sample were adjusted to 0.15 3 106 sperm/mL in human tubal fluid (HTF; Irvine Scientific, Irvine, CA) and 8% maternal serum for IVF. Higher concentrations, 0.3 3 106 628
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IVF and ICSI in subfertile males
sperm/mL, were used in some cases when semen parameters were poor. The tube was capped loosely and set in the incubator at 37°C, 5% CO2 in air until the time of insemination. The sample for ICSI was prepared by the addition of 0.3 mL of modified sperm wash media (MSWM; Irvine Scientific) to the pellet. The tube was tightly capped and kept at 37°C until the time of sperm injection.
Oocyte Preparation and Insemination Oocytes were assigned randomly to either IVF or ICSI. The IVF oocytes were kept in 100-mL drops of HTF and 8% maternal serum. Five to six hours postaspiration, one oocyte was placed in 15,000 sperm per 100-mL drop. The ICSI oocytes were placed in hyaluronidase to remove the cumulus, rinsed in HTF and maternal serum and stripped of the corona cells. Only oocytes with a polar body were injected. Several injection dishes were prepared with one drop of polyvinyl pyrrolidone (Irvine Scientific) and one drop of MSWM. Each oocyte was injected with sperm of normal morphology displaying motility on Nikon-Narishige micromanipulation equipment (Melville, NY) using injection and holding pipettes from Humagen Fertility Diagnostics (Charlottesville, VA). Once injection was completed, the oocytes were rinsed in HTF and maternal serum.
Fertilization and Embryo Quality On day 1 (16 –20 hours after retrieval) the oocytes were checked for fertilization. The IVF oocytes were stripped to remove the corona. The presence of more than 2 pronuclei (2PN) was noted. Oocytes with 2PN were transferred to growth media (HTF 1 15% maternal serum). On day 2 (40 – 43 hours after retrieval), embryo quality was recorded by cell number and cell grade. Cell grade was classified according to the following modified D. P. Wolf criteria (10). Grade 4 and 5 embryos are considered superior quality embryos, with the blastomeres evenly shaped and equal in size. Grade 5 embryos contain no cytoplasmic fragments, and grade 4 embryos have cytoplasmic fragments consisting of ,10% of the embryo volume. Grade 3 embryos have blastomeres with slightly distorted shapes and sizes that are slightly uneven. Cytoplasmic fragments may be present in up to 25% of the embryo volume. Grade 2 embryos contain significantly distorted blastomeres that are unequal in size, and the cytoplasmic fragments make up 25%–50% of the embryo volume. The poorest quality embryos, grade 1, have uneven fragmented blastomeres with .50% of the embryo volume consisting of cytoplasmic fragments. The ooplasm is dark and flat, distinct from the nongranular and nonstatic ooplasm of the other grades. For the purposes of this study, a scoring system was developed incorporating cell number and cell grade (Table 1).
Embryo Transfer After oocyte aspiration, prednisone (10 mg b.i.d.) and tetracycline (250 mg q.i.d.) was administered for 4 days. Vol. 71, No. 4, April 1999
TABLE 1
TABLE 2
Embryo quality scoring system. Cell no.
Comparison of IVF and ICSI from sibling oocytes in subfertile males.
Cell grade*
Score
5 4 5 4 5 4 5 4 5 4 3 3 3 3 3 2 2 2 2 2 1
1 Best quality 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Poorest quality
Treatment 6 6 5 5 4 4 3 3 2 2 6 5 4 3 2 6 5 4 3 2 Any cell number 2PN Note: PN 5 pronuclei. * Modified D. P. Wolf criteria.
Progesterone in oil 25 mg (50 mg/mL; Steris Laboratories, Phoenix, AZ) was given for daily luteal support starting the day of ET and continued until menses or 8 gestational weeks. Up to six embryos were transferred with use of a Wallace catheter.
Pregnancy Assessment
Serum b-hCG was obtained 14 days after aspiration, and confirmatory ultrasound was performed 2 weeks later. Clinical pregnancy was defined as the presence of a sac at 5 weeks of gestation.
Statistical Analysis Fisher’s exact test was used to compare the number of successful cycles with IVF versus ICSI. The oocyte fertilization rate was calculated as the number of fertilized oocytes divided by the number of metaphase II oocytes inseminated. The overall number of oocytes fertilized with IVF versus ICSI was compared with use of the x2 test, and percentage of oocytes fertilized per couple were compared between IVF and ICSI by the Wilcoxon matched-pairs test. Embryo quality, based on cell number, grade, and score was compared with the Mann-Whitney U-test, and the x2 test was used to compare superior quality embryos (based on grade and score) between IVF and ICSI. This protocol was approved by the Institutional Review Board at Baylor College of Medicine and affiliated hospitals. FERTILITY & STERILITYt
Variable No. of cycles with fertilization/total no. of cycles (%) No. of oocytes fertilized/no. of oocytes allocated (%) Mean (6SEM) percentage of oocytes fertilized No. of grade 4 or 5 embryos/total no. of embryos (%) * † ‡ §
P P P P
5 5 5 5
not not not not
significant significant significant significant
(determined (determined (determined (determined
by by by by
IVF
ICSI
13/18 (72)*
17/18 (94)*
69/120 (58)†
80/131 (61)†
44.6 6 9.0‡
62.7 6 5.6‡
32/69 (46)§
38/74 (51)§
Fisher’s exact test). x2-test). Wilcoxon matched-pairs test). x2-test).
RESULTS Subfertile Male Group (Group A) A total of 18 couples with one or more abnormal semen parameters underwent an IVF cycle in which oocytes were divided between conventional IVF and ICSI (Table 2). Fertilization occurred in 13 of 18 (72%) of cycles with IVF and 17 of 18 (94%) of cycles with ICSI (P5.18). The single cycle with failed fertilization after ICSI also had failed fertilization with conventional IVF. A total of 120 metaphase II oocytes were allocated to IVF, and 131 metaphase II oocytes were allocated to ICSI. Overall, 69 of 120 (58%) of the oocytes fertilized with IVF, whereas 80 of 131 (61%) fertilized with ICSI (P 5 .57). Of the total cohort injected, the damage rate was 17 of 131 (13%). The mean (6SEM) percentage of oocytes fertilized per couple was 44.6% 6 9.0% with IVF and 62.7% 6 5.6% with ICSI (P 5 .09). Of the 18 couples, 7 had better fertilization with IVF than ICSI. Comparisons between percent fertilization and semen parameters were made. There was no correlation between fertilization rates per couple with either IVF or ICSI and sperm count, motility, or motile sperm concentration. There was no statistically significant difference in embryo quality based on cell number, cell grade, and score between embryos from IVF versus ICSI. Thirty-two of the 69 (46%) embryos from IVF were either grade 4 or 5 (equivalent to a score of #10). Similarly, 38 of 74 (51%) of embryos from ICSI were grade 4 or 5. This was also not statistically significant. Differences in pregnancy rates with IVF versus ICSI could not be assessed because embryos were transferred concurrently from both procedures. Eleven of the 18 (61%) couples achieved pregnancy. Currently, there are four ongoing intrauterine pregnancies, seven delivered intrauterine 629
DISCUSSION
TABLE 3 Comparison of IVF and ICSI from sibling oocytes in males with severe teratozoospermia. Treatment Variable No. of cycles with fertilization/total no. of cycles (%) No. of oocytes fertilized/no. of oocytes allocated (%) Mean (6SEM) percentage of oocytes fertilized No. of grade 4 or 5 embryos/total no. of embryos (%) * † ‡ §
IVF
ICSI
18/20 (90)*
20/20 (100)*
54/113 (48)†
82/124 (66)†
50.9 6 7.1‡
66.6 6 4.7‡
16/54 (30)§
24/79 (30)§
P 5 not significant (determined by Fisher’s exact test). P5.004 (determined by x2-test). P5.04 (determined by Wilcoxon matched-pairs test). P 5 not significant (determined by x2-test).
pregnancies, and one second trimester loss secondary to chorioamnionitis. There were seven multiple gestations. Two intrauterine pregnancies (1 set of term twins and the second trimester twin loss) were achieved in couples who had fertilization with ICSI only.
Teratozoospermia Group (Group B) Twenty couples for whom severe teratozoospermia (morphology of #4% normal forms) was the only identifiable cause of subfertility were separately analyzed (Table 3). Fertilization occurred in 18 of 20 (90%) cycles with IVF and 20 of 20 (100%) cycles with ICSI (P5.49). A total of 113 metaphase II oocytes were allocated to IVF, and 124 metaphase II oocytes were allocated to ICSI. Overall, 54 of 113 (48%) of the oocytes fertilized with IVF, whereas 82 of 124 (66%) fertilized with ICSI (P5.004). Of the total cohort injected, the damage rate was 17 of 124 (14%). The mean (6SEM) percentage of oocytes fertilized per couple was 50.9% 6 7.1% with IVF and 66.6% 6 4.7% with ICSI (P5.04). There was a statistically significant difference in the overall number of oocytes fertilized with IVF versus ICSI and the percentage of oocytes fertilized per couple. There was no statistically significant difference in embryo quality based on cell number, cell grade, and score between embryos from IVF versus ICSI. Sixteen of the 54 (30%) embryos from IVF were either grade 4 or 5 (equivalent to a score of #10), and 24 of 79 (30%) of embryos from ICSI were grade 4 or 5. Seven of the 20 (35%) couples achieved pregnancy. Currently, there are five ongoing singleton pregnancies, one term infant, and one ectopic pregnancy—an intrauterine pregnancy rate of 30%. One ongoing pregnancy was achieved in a couple who had fertilization with ICSI only. 630
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IVF and ICSI in subfertile males
In men whose only semen abnormality was severe teratozoospermia, ICSI resulted in a statistically superior overall number of oocytes fertilized and percentage of oocytes fertilized per couple, compared with conventional IVF, without altering embryo quality. However, ICSI did not significantly improve fertilization or embryo quality compared with conventional IVF of sibling oocytes in this population of subfertile males, defined by the presence of at least one abnormal semen parameter (count of ,20 3 106/mL or motility of ,40% [WHO]) regardless of morphology (Kruger’s strict criteria [7, 9]). By using sibling oocytes, we were able to remove any female factor that might affect fertility, resulting in a more accurate evaluation of IVF versus ICSI in this infertile population. The number of oocytes fertilized per couple between IVF and ICSI constitutes the strength of this study and was not performed in previous sibling oocyte comparisons. There was no significant difference after ICSI in the number of cycles with fertilization, overall number of oocytes fertilized, and percent oocytes fertilized per couple, in this population of subfertile males. However, only 1 couple of 18 had failed fertilization with ICSI, whereas 5 of 18 couples had failed fertilization with IVF. Further evaluation of this population did not reveal any predictors of poor outcome. We did not demonstrate the same statistically increased overall number of oocytes fertilized with ICSI compared with conventional IVF in a subfertile male population as did Aboulghar et al. (5). In the former study, also using sibling oocytes, subfertile males were defined as having at least one normal semen analysis based on WHO criteria and at least one subnormal parameter on another semen analysis. In this group of 24 couples, the overall number of oocytes fertilized with conventional IVF was 27.1% and 59% with ICSI. However, these investigators did not pair the data to statistically analyze fertilization in each couple, the strength of using sibling oocytes. The most important semen parameter that affected fertilization when comparing conventional IVF with ICSI was morphology in the presence of normal count and motility. In couples with severe teratozoospermia, #4% normal forms by Kruger’s strict criteria, ICSI increased the number of oocytes fertilized by 18% (P5.004) compared with IVF, and the fertilization rate per couple was increased from 50.9% to 66.6% (P5.04). It is surprising that only two couples had complete fertilization failure with conventional IVF, and none of the couples had failed fertilization with ICSI. This is consistent with the widely held tenet that morphology is one of the most important semen parameters predicting fertilization in conventional IVF. Initially, the overall number of oocytes fertilized was as low as 7.6% in Vol. 71, No. 4, April 1999
patients with severe teratozoospermia (7). Improvements in IVF has led to higher numbers of oocytes fertilized in couples with severe teratozoospermia, but these rates still remain significantly lower than in couples with a strict morphology of .4% (8, 11). Ombelet et al. (11) assessed IVF success based on sperm morphology. They found that the overall number of oocytes fertilized in couples with ,9% normal forms, based on Kruger’s strict criteria, was 69.2% compared with 79.4% in couples with .9% normal forms. Ombelet et al. (11) further divided these patients with ,9% normal forms into a poor prognosis group with ,5% normal forms based on a fertilization rate per oocyte of 37.9% with a 0% pregnancy rate and a good prognosis group, 5%– 8% normal forms, with a fertilization rate per oocyte of 78.6% and a 15.8% pregnancy rate. However, this group did not use sibling oocytes. Unlike IVF, ICSI is not dependent on any semen parameters (3, 12) including morphology. Mansour et al. (13) found no significant difference in fertilization and pregnancy rates between patients with ,5% normal forms using strict criteria with a 59% fertilization rate per oocyte and 36.6% pregnancy rate and patients with .5% normal forms with a 57.3% fertilization rate per oocyte and a pregnancy rate of 34.8%. Again, no sibling oocytes were used. Although teratozoospermia has been associated with poor fertilization using conventional IVF, whereas fertilization with ICSI is not affected routinely, none of these studies used sibling oocytes to account for uncontrollable differences between groups. Only one other study evaluated “conventional” IVF with ICSI in sibling oocytes in men with poor morphology (2). Unlike our study, poor morphology was defined as ,20% normal forms based on WHO criteria. Furthermore, poor semen morphology was not always the only abnormal semen parameter. This may account for the study’s extremely poor overall fertilization rate per oocyte of 15%, with only 39% of cycles with fertilization, using conventional IVF compared with an overall fertilization rate per oocyte of 76%, with 100% of cycles with fertilization, when ICSI was used. Similar findings were observed in studies, using sibling oocytes, when conventional IVF was replaced with high insemination concentration IVF. Fishel et al. (14) found that 37% of oocytes fertilized with high insemination concentration IVF versus 58% with ICSI in patients with ,5% normal forms. The number of oocytes fertilized in patients with .5% normal forms were equivalent whether high insemination concentration IVF or ICSI was used. However, Fishel’s patient population failed prior conventional IVF, either on two previous occasions or on a single occasion in which there was no binding to the zona pellucida. This group was even more selective than the group in our study and may represent the subpopulations in our group that benefit using ICSI. Nonetheless, it would be impractical to FERTILITY & STERILITYt
let patients fail one IVF cycle before subjecting them to IVF and ICSI. Only one study found superior fertilization rates per oocyte with high insemination concentration IVF over ICSI, 86% versus 68%, respectively. Unfortunately, this study did not evaluate sibling oocytes (15). Again, even in those studies mentioned above, no paired statistical analysis was performed to assess fertilization within each couple. We could not compare pregnancy rates as a result of IVF versus ICSI because couples in our study had embryos transferred from both procedures. However, we did not find any difference in embryo quality between IVF fertilized embryos and ICSI embryos in either group, the subfertile males or the teratozoospermic males. Embryo quality was based on a scoring system incorporating cell number and cell grade using modified D. P. Wolf criteria (10). One other study found similar results when oocytes were divided between high insemination concentration IVF and ICSI in couples with severe teratozoospermia (16). In contrast, Oehninger et al. (15) found that ICSI produced superior quality embryos, defined as grade 1 or 2, in couples with severe teratozoospermia, than with high insemination concentration IVF, 87% grade 1 and 2 embryos with ICSI compared with 72% with high insemination concentration IVF. Similarly, Fishel et al. (14) also found that in couples with severe teratozoospermia, high insemination concentration IVF resulted in compromised cleavage morphology over ICSI, only 30% of embryos after high insemination concentration IVF were normal compared with 84% with ICSI. However, these couples had a history of failed fertilization with conventional IVF and may represent a different subpopulation. Although counterintuitive, the embryo quality may be the result of the higher concentration of sperm decreasing embryo quality rather than ICSI improving it. In studies comparing IVF with ICSI in nonmale factor infertility, Yang et al. (6) found statistically more superior quality embryos, defined as grade A embryos, in the ICSI group than in the IVF group, 35.4% versus 24.3%, respectively. Yang et al. (6) explained the improved embryo quality because “ICSI technique avoids oocytes and zygotes cultured with a lot of spermatozoa (100,000 –500,000 spermatozoa per well), which does not happen in vivo. Human spermatozoa can produce reactive oxygen species such as superoxide anion (O22) and hydrogen peroxide (H2O2) (17, 18). It seems likely that free radical toxicity also contributes to embryonic damage during the culture period necessary for human IVF.” If free radical toxicity does contribute to embryonic damage, a smaller concentration of spermatozoa should be used, paradoxically, to improve embryo quality with IVF. Although there is a trend toward improved fertilization with ICSI in this specific population of subfertile males with low semen parameters, other than morphology, there is no 631
single parameter that may act as a predictor to determine outcome. Until such a predictor becomes available, based on our results, conventional IVF is an option in this population without the disadvantages of ICSI, including higher costs, increased time consumption, and personnel training. However, with increased cycles of failed fertilization with IVF compared with ICSI in this subpopulation, the desire to optimize chances of pregnancy may lead to ICSI as the preferred treatment option in this group of patients. One semen parameter that may act as a predictor for using ICSI as a first-line therapeutic modality is morphology. Severe teratozoospermia, defined as #4% normal forms according to Kruger’s strict criteria (7, 9), is the most significant factor that affects fertilization when IVF is used. Thus, ICSI should be used in this patient population with subsequent high fertilization rates and good embryo quality. References 1. Society for Assisted Reproductive Technology and the American Society of Reproductive Medicine. Assisted reproductive technology in the United States and Canada: 1995 results generated from the american society for reproductive medicine/society for assisted reproductive technology registry. Fertil Steril 1998;69:389 –98. 2. Payne D, Flaherty SP, Jeffrey R, Warnes GM, Matthews CD. Successful treatment of severe male factor infertility in 100 consecutive cycles using intracytoplasmic sperm injection. Hum Reprod 1994;9:2051–7. 3. Palermo G, Joris H, Derde MP, Camus M, Devroey P, Van Steirteghem AC. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil Steril 1993;59:826 –35. 4. Van Steirteghem AC, Liu J, Joris H, Nagy Z, Janssenswillen C, Tournaye H, et al. Higher success rate by intracytoplasmic sperm injection than by subzonal insemination: report of a second series of 300 consecutive treatment cycles. Hum Reprod 1993;8:1055– 60. 5. Aboulghar MA, Mansour RT, Serour GI, Sattar MA, Amin YM. Intracytoplasmic sperm injection and conventional in vitro fertilization for sibling oocytes in cases of unexplained infertility and borderline semen. J Assist Reprod Genet 1996;13:38 – 42.
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6. Yang D, Shahata MA, Al-Bader M, Al-Natsha SD, Al-Flamerzia M, Al-Shawaf T. Intracytoplasmic sperm injection improving embryo quality: comparison of sibling oocytes of non-male-factor couples. J Assist Reprod Genet 1996;13:351–5. 7. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1988;49:112–7. 8. Grow DR, Oehninger S, Seltman HJ, Toner JP, Swanson RJ, Kruger TF, et al. Sperm morphology as diagnosed by strict criteria: probing the impact of teratozoospermia on fertilization rate and pregnancy outcome in a larger in vitro fertilization population. Fertil Steril 1994;62:559 – 67. 9. Menkveld R, Stander FSH, Kotze TJ, Kruger TF, van Zyl JA. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 1990;5:586 –92. 10. Wolf DP, ed. In vitro fertilization and embryo transfer, a manual of basic techniques. New York: Plenum Press, 1988. 11. Ombelet W, Fourie FL, Vandeput H, Bosmans E, Cox A, Janssen M, et al. Teratozoospermia and in vitro fertilization: a randomized prospective study. Hum Reprod 1994;9:1479 – 84. 12. Cohen J, Talansky BE, Malter H, Alikani M, Adler A, Reing A, et al. Microsurgical fertilization and teratozoospermia. Hum Reprod 1991;6: 118 –23. 13. Mansour RT, Aboulghar MA, Serour GI, Amin YM, Ramzi AM. The effect of sperm parameters on the outcome of intracytoplasmic sperm injection. Fertil Steril 1995;64:982– 6. 14. Fishel S, Lisi F, Rinaldi L, Lisi R, Timson J, Green S, et al. Intracytoplasmic sperm injection (ICSI) versus high insemination concentration (HIC) for human conception in vitro. Reprod Fertil Dev 1995;7: 169 –75. 15. Oehninger S, Kruger TF, Simon T, Jones D, Mayer J, Lanzendorf S, et al. A comparative analysis of embryo implantation potential in patients with severe teratozoospermia undergoing in-vitro fertilization with a high insemination concentration or intracytoplasmic sperm injection. Hum Reprod 1996;11:1086 –9. 16. Hall J, Fishel S, Green S, Fleming S, Hunter A, Stoddart N, et al. Intracytoplasmic sperm injection versus high insemination concentration in-vitro fertilization in cases of very severe teratozoospermia. Hum Reprod 1995;2:493– 6. 17. Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa: superoxide dismutase as a major enzyme protectant against oxygen toxicity. J Androl 1987;8:338 – 48. 18. Atiken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 1987;81:459 – 69.
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