In vitro fertilization outcome in the presence of severe male factor infertility*

Transcription

1 FERTILITY AND STERILITY Vol. 63, No.5, May 1995 Copyright 1995 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. In vitro fertilization outcome in the presence of severe male factor infertility* Avraham Ben-Chetrit, M.D.t Selim Senoz, M.D. Ellen M. Greenblatt, M.D. Robert F. Casper, M.D.:!: Division of Reproductive Sciences, Department of Obstetrics and Gynecology, The Toronto Hospital, Toronto, Ontario, Canada Objective: To assess the outcome of standard IVF treatment (nonmicromanipulated) with respect to total motile sperm number recovered by swim-up, particularly for couples with severe male factor infertility defined as total motile sperm number < 0.5 X 10 6 Design: Retrospective study of patients who underwent successful oocyte retrieval in an IVF program from August 10, 1992 to December 31, Setting: A university-based tertiary referral center (The Toronto Hospital). Patients: All cycles (n = 672) were divided into four groups according to total motile sperm number recovered using standard swim-up: group 1, total motile sperm number : X 10 6 ; group 2, total motile sperm number between 0.51 and 1.00 X 10 6 ; group 3, total motile sperm number between 1.01 and 1.50 X 10 6 ; and group 4, total motile sperm number 2: 1.51 X 10 6 All patients received the same controlled ovarian hyperstimulation protocol, which consisted of a GnRH analog flare-up followed by parenteral menotropins. Clinical and cycle characteristics in the four groups were analyzed and outcome was evaluated. Results: There was no significant difference in clinical and cycle characteristics between the groups. The uniformity of the groups justified analysis of their outcome. A fertilization rate of 21.5% was achieved in couples with severe male factor (group 1). Fertilization rate and number of embryos transferred increased directly with the total motile sperm number. There was no significant difference in implantation rate per embryo between the groups. Conclusions: The results in couples with severe male factor infertility compare favorably with monospermic fertilization rates reported in the literature using partial zona dissection and subzonal insertion but is lower than with intracytoplasmic sperm injection. Therefore, we believe that couples with severe male factor infertility should be considered for standard IVF, as long as adequate total motile sperm can be recovered (100 X 10 3 per dish). Ifintracytoplasmic sperm injection is available, it should be offered to these couples. Fertil Steril 1995;63: Key Words: In vitro fertilization, male factor, implantation rate, micromanipulation The crucial role of sperm quality in human reproduction is well established (1) and is highlighted by Received June 13, 1994; revised and accepted December 5, * Supported by The Medical Research Council of Canada, Ottawa, Ontario, Canada. t Supported by a fellowship grant from The Schiff Family Foundation, Toronto, and Serono, Mississauga, Ontario, Canada. :j: Reprint requests: Robert Casper, M.D., EN, The Toronto Hospital, 200 Elizabeth Street, Toronto, Ontario, M5G 2C4 Canada (FAX: ). the introduction of assisted reproductive technologies whereby the capability of spermatozoa to fertilize the oocyte can be assessed directly. In vitro fertilization programs report lower pregnancy rates after lower fertilization rates when insemination is performed with subnormal spermatozoa (2,3). The term severe male factor is used widely, but a clear definition and prognosis for men with this diagnosis has not been established. Debate exists concerning the importance of each semen parameter and its contribution to fertilization in standard IVF. Although 1032 Ben-Chetrit et at. NF outcome in severe male factor Fertility and Sterility

2 some studies have found morphology (4,5), motility (2), or sperm count (6) most predictive for IVF outcome, many laboratories accept total motile sperm count (after semen preparation) as the most accurate predictive parameter of fertilizing ability (3, 7-9). It has been reported that no fertilization occurred when the concentration of motile spermatozoa was <1 X 106/ml (10) or when the number of total motile spermatozoa was <1.5 X 10 6 (3). Furthermore, although adequate numbers of spermatozoa for IVF can be obtained from these samples, no improvement in fertilization rate has been detected with the use of higher than the normal insemination concentrations (3). These findings suggest that the pathology leading to low total motile sperm number also may affect the capability of spermatozoa to fertilize. However, once fertilization occurs, the probability of implant a tion for each embryo appears to be the same, regardless of sperm quality (3, 11). To overcome the low fertilization rate associated with severe male factor infertility, micromanipulation techniques have been developed, and many infertile couples with severe male factor have been treated by these methods (12, 13). Unfortunately, few laboratories have the resources, both in equipment and trained personnel, to offer micromanipulation. Furthermore, not all methods of micromanipulation for severe male factor infertility are associated with high monospermic fertilization rate (9, 12, 14-19). The objectives of our study were to assess the value of standard IVF for couples with severe male factor «0.5 x 10 6 motile spermatozoa after swim up) and to compare monospermic fertilization rates with those published for the various micromanipulation techniques MATERIALS AND METHODS A retrospective study was performed of 672 cycles of successful oocyte retrieval in the University of Toronto based IVF program (The Toronto Hospital, Toronto, Ontario, Canada) from August 10, 1992 to December 31, All cycles were divided into four groups according to the total motile sperm number recovered from the swim-up sample: group 1, cycles whose total motile sperm number was $0.50 X 10 6 and defined as severe male factor infertility; group 2, cycles whose total motile sperm number ranged from 0.51 to 1.00 x 10 6 and defined as moderate male factor infertility; group 3, cycles whose total motile sperm number ranged from 1.01 to 1.50 x 10 6 and defined as mild or borderline male factor infertility; and group 4, cycles whose total motile sperm number was :;;::1.51 x 10 6 and defined as normal. Ovarian Stimulation and Ovum Retrieval All patients were on oral contraceptives (OCs) for 18 to 35 days before their IVF cycle to allow schedul- Vol. 63, No.5, May 1995 ing of oocyte retrieval. Controlled ovarian hyperstimulation consisted of a flare-up protocol, using daily SC GnRH analog (1 mg leuprolide acetate; Lupron; Abbott Pharmaceutical Company Ltd., Pointe Claire, Quebec, Canada) and parenteral gonadotropins beginning on the 5th day after an OC-induced withdrawal bleed. The standard protocol began with two ampules of hmg (75 IU per ampule; Pergonal; Serono, Mississauga, Ontario, Canada) in women < 35 years and three ampules in women> 35 years. Daily ultrasound for follicular measurement was performed starting on the 3rd day of gonadotropin administration. Blood samples for measurement of serum E2 concentration were drawn daily. Human chorionic gonadotropin (5,000 IV; Profasi; Serono) was administered when the mean diameter of at least two follicles measured :;;:: 18 mm and the E2 level approximated 1,000 pmol per mature follicle. Transvaginal ultrasound -guided ovum retrieval was performed 36 hours after hcg administration. Semen Preparation and Insemination Semen samples were collected on the morning of oocyte retrieval. Mter 30 minutes of liquefaction at room temperature, the sample was diluted with 3 volumes of human tubal fluid medium supplemented with 10% human serum albumin. The diluted semen was centrifuged for 10 minutes at 500 X g, the supernatant was discarded, and the pellet was resuspended in 2 ml medium. The suspension was centrifuged again under the same conditions, and the supernatant was discarded. The pellet was resuspended in 200 ml of medium, aspirated, and layered gently under 1 ml of fresh medium. Motile spermatozoa were allowed to swim up into the overlaying medium at 37 C in a 5% CO 2, 5% O2, and 90% N2 atmosphere. When the overlaying medium became cloudy (usually at the end of 1 hour), it was aspirated, and the sperm concentration, motility, morphology, and forward progression were evaluated. Total motile sperm count was calculated as the product of sperm concentration, volume, and percent motility. Four to 5 hours after oocyte retrieval, one to four oocytes in each dish were inseminated with 100,000 motile sperm. In all cycles, this minimal number of sperm always was available. Therefore, the number of sperm added to each dish did not compromise the potential of the oocytes to be fertilized. Sixteen to 20 hours later, the oocytes were examined for the presence of two pronuclei. Embryo Transfer and Luteal Phase Support Forty-eight hours after oocyte retrieval, up to three embryos were transferred. Freezing of extra Ben-Chetrit et ai. NF outcome in severe male factor 1033

3 Table 1 In Vitro Fertilization Outcome in Cycles With Successful Retrieval Group Total motile sperm number (x10 6 ) No. of patients Fertilization rate (%)* No. of embryos transferred* Pregnancy rate per retrieval~** so ::': 4.7t 0.9::': 0.2 3/38 (7.8) 0.51 to ::': ::': (14.8) 1.01 to ::': ::': /47 (21.2) s ::': ::': /513 (22.4) * Values are means::': SEM. t Group 1 versus groups 2, 3, and 4, P < Group 2 versus groups 3 and 4. Group 1 versus groups 2, 3, and 4, P < II Group 2 versus groups 3 and 4. ~ Values in parentheses are percentages. ** P < fertilized oocytes was offered to all patients. Luteal support consisted of hcg (1,500 IU Profasi), every 3rd day for 2 weeks, accompanied by P vaginal suppositories. One week after the last hcg injection, the serum,b-hcg level was measured. If the,b-hcg was positive, the patient was scheduled 2 weeks later for transvaginal ultrasound screening for presence of clinical pregnancy as documented by the visualization of an intrauterine gestational sac. Clinical Evaluation Clinical and cycle characteristics evaluated included patient age, duration of OC use before ovulation induction, number of hmg ampules used for ovarian hyperstimulation, cycle day ofhcg administration, E2 level and endometrium thickness on day of hcg administration, and the number of oocytes retrieved per patient. Although group 1 appeared to be slightly younger than the other three groups (P = 0.03), it is unlikely that this small statistical difference was clinically significant. The rest of the clinical characteristics were very close and not significantly different between the four groups. Fifty percent of the cases in group 1 and 67% in group 2 had male factor as a primary diagnosis as well as another female diagnosis. The distribution of female diagnosis was similar to the general infertility population, and it should not skew the IVF outcome. Outcome parameters included fertilization rate (number of embryos obtained divided by number of oocytes inseminated), number of embryos transferred, clinical pregnancy rate (at least one gestational sac identified on ultrasound), and implantation rate (number of gestational sacs divided by number of embryos transferred). Statistical Evaluation SPSS PC( + ) (SPSS Inc., Chicago, IL) was used for analysis. Data were analyzed by one-way analysis of variance and X 2 where appropriate. A difference 1034 Ben-Chetrit et al. IVF outcome in severe male factor of P < 0.05 was considered to be significant. All values are given as mean ± SEM. RESULTS Table 1 demonstrates the gradual increase in fertilization rate, which was correlated directly with total motile sperm number. Consequently, the increasing total motile sperm number was associated with an increased number of embryos as well as the number of embryos transferred (only groups 3 and 4 were not different significantly from each other). Although not significant, the pregnancy rate per retrieval showed an increasing trend correlated with total motile sperm number. Most importantly, even in group 1 (with severe male factor infertility), a reasonable pregnancy rate of 7.8% was achieved. When we examined only the cycles in which at least one oocyte fertilized (Table 2) we observed a significant increase in fertilization rate and the number of embryos transferred, which was correlated with total motile sperm number. Although the pregnancy rate per ET showed an increasing trend, it was not significantly different. Moreover, as expected (3, 7), once fertilization occurred, the implantation rate was not affected and further embryo development appeared to be independent of semen characteristics. Failure to fertilize occurred in 47.3% of group 1 cycles (Table 2) versus only 10.1% of group 4 cycles. This gradual decrease in failure to fertilize correlates significantly with the increase in total motile count. DISCUSSION In the last decade, IVF has been recognized as an important treatment for infertile couples with male factor infertility (7). However most IVF centers report unfavorable results in cases with severe male factor. It is clear that the ultimate test to evaluate the sperm's capability to fertilize remains an IVF Fertility and Sterility

4 Table 2 In Vitro Fertilization Outcome in Relation to Fertilization Group Total motile sperm number (x10 6 ) < to to 1.50 >1.51 No. of patients with fertilization of at least one oocyte Fertilization in couples with at least one embryo (%)* 40.8 ± 6.4t 56.5 ± 4.2:j: 71.8 ± ± 1.1 No. of embryos transferred 1.8 ± ± ± ± 0.04 Pregnancy rate per ET~** 3/20 (15) (21.5) 10/39 (25.6) 115/461 (24.9) Implantation rate (%)tt 7.9 ± ± ± ± 1.1 No. of patients with no fertilization Failed fertilization~:j::j: 18/38 (47.3) 23/74 (31.0) 8/47(17) 52/513 (10.1) * Values are means ± SEM. t Group 1 versus groups 2, 3, and 4, P < :j: Group 2 versus groups 2, 3, and 4, P < Group 1 versus groups 2, 3, and 4, P < II Group 2 versus groups 2, 3, and 4, P < ~ Values in parentheses are percentages. ** Not statistically significantly different, P = 0.7. tt Not statistically significantly different, P = 0.8. :j::j: Groups 1 and 2 versus groups 3 and 4, P < trial. However, a definitive semen parameter that can be an accurate predictor for oocyte fertilization remains elusive. Although some studies show higher predictive value for morphology (4, 5), others emphasize the importance of motility (2) or sperm count (6). Like others (3, 7-9), we found that total motile count is a reasonably reliable parameter for prediction of IVF outcome. Because it has been reported that no fertilization occurred when the concentration of motile spermatozoa was < 1 X 10 6 /ml (10) or when the number of total motile spermatozoa was < 1.5 X 10 6 (3), IVF centers often limit acceptance of couples by threshold values of total motile sperm number after sperm preparation by swim-up or Percoll gradient centrifugation. This total motile sperm number threshold has ranged from 18 X 10 6 (20) through 5 X 10 6 (21) to as low as 1.5 X 10 6 (22). In this study, we found a low but significant fertilization rate of21.1 % even in the group with <0.5 X 10 6 total motile spermatozoa. This result supports the finding reported by Ord et al. (8) that 25% of a group in which the average total motile sperm number was 0.72 X 10 6 were able to fertilize their partners' eggs. The pregnancy rate of 7.8% achieved in group 1 (total motile sperm number :s; 0.5 X 10 6 motile sperm) and the implantation rate of 7.9% per embryo indicate that embryos that develop in couples with severe male factor infertility have the same developmental potential as other diagnostic categories of infertility. The higher pregnancy rate in the other groups likely reflects the increased number of embryos transferred (23). The reported monospermic fertilization rates in partial zona dissection range from 10.1% to 27.7% (12, 14, 15, 17), whereas those of subzonal insemination (SUZI) range from 16% to 20.4% (9, 14, 16, 18, 19). These results do not appear to be significantly higher than the rates reported using standard IVF Table 3 Monospermic Fertilization Rates in Micromanipulated IVF Compared With Standard IVF in Cases of Severe Male Factor Infertility No. of oocytes No. of oocytes Fertilization Fertilizing method treated fertilized rate Fishel et al. (9) SUZI 1, Catt et al. (16) SUZI 2, Cohen et al. (14) SUZI Sakkas et al. (18) Center 1 SUZI Center 2 SUZI Van Steirteghem et al. (19) SUZI 1, Cohen et al. (14) Partial zona dissection Gordon et al. (12) Partial zona dissection Vanderzwalmen et al. (17) Partial zona dissection Calderon et al. (15) Partial zona dissection 1, Van Steirteghem et al. (24) ICSI 1, Ord et al. (8) Standard IVF Present study Standard IVF % Vol. 63, No.5, May 1995 Ben-Chetrit et al. NF outcome in severe male factor 1035

5 (Table 3). However, with the development of in tracytoplasmic sperm injection, the reported fertilization rate of >60% is significantly higher than standard IVF. In two consecutive publications, Van Steirteghem et al. (19, 24) report the intracytoplasmic sperm injection fertilization rate in severe male factor cases to range from 63.2% to 69.9%. Therefore, although almost half of the patients with <0.5 X 10 6 motile spermatozoa had no fertilization (Table 2), as long as it is possible to recover an adequate number of motile spermatozoa for insemination (100 X 10 3 per dish), we suggest that a standard IVF trial is indicated. Furthermore, it may be reasonable, as suggested by Ben-Shlomo et al. (25), to try two IVF cycles before considering partial zona dissection or SUZI. However, if available, referral of patients with severe male factor to centers where intracytoplasmic sperm injection is offered would appear to give the highest chance of success in this group. Couples with moderate male factor infertility according to our definition (group 2) showed a significant increase in fertilization rate and number of embryos transferred compared with group 1. A fertilization rate of 39.8% in this group does not justify offering these couples partial zona dissection or SUZI. However, intracytoplasmic sperm injection still may have a superior fertilization rate, and this method should be considered when counseling couples in this category. Couples with mild or borderline male factor (group 3) have similar IVF outcomes to normals (group 4), and both have significantly better fertilization rates than group 1 and 2. Having fertilization rates of approximately 60%, these groups should not be considered as candidates for any micromanipulation procedure. In conclusion, couples with severe male factor infertility, even when total motile sperm number is <0.5 X 10 6, should be offered a trial of conventional IVF, as long as an adequate number of motile spermatozoa can be recovered for insemination. Although more studies are needed to confirm the fertilization and pregnancy rates reported, we believe that an intracytoplasmic sperm injection program, if available, should be considered as the best possible option for couples with severe male factor infertility. In the absence of an intracytoplasmic sperm injection program, partial zona dissection and SUZI should be considered in cases where the number of motile sperm recovered is less than needed for insemination in routine IVF or in cases from any group that demonstrates fertilization failure in two or more IVF trials. Acknowledgment. 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