FERTILITY AND STERILITY Copyright e 1988 The American Fertility Society Printed in U.S.A. Corrective measures and pregnancy outcome in in vitro fertilization in patients with severe sperm morphology abnormalities Sergio Oehninger, M.D.* Anibal A. Acosta, M.D. Mahmood Morshedi, Ph.D. Lucinda Veeck, M.L.T. R. James Swanson, Ph.D. Kathryn Simmons, M.S. Zev Rosenwaks, M.D. Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia Sperm morphology evaluated by new, strict criteria is a good predictor of outcome in in vitro fertilization (IVF). This study aimed (1) to determine whether the fertilization rate of preovulatory oocytes in patients with abnormal morphology can be improved by increasing insemination concentration at the time of IVF and (2) to evaluate the pregnancy outcome in patients with abnormal sperm morphology. Three groups were studied: (1) normal morphology, (2) good prognosis pattern, and (3) poor prognosis pattern. other sperm parameters were normal. Group 3 had a lower overall fertilization rate, lower pregnancy rate/cycle, and lower ongoing pregnancy rate/cycle. Groups 2 and 3 showed a higher miscarriage rate, although not significantly different from group 1. By increasing insemination concentration from 2- to 10-fold, the fertilization rate in group 3 increased from 14.5% to 62.6%. However, pregnancy outcome did not improve. We conclude that patients with severe sperm head abnormalities have a lower ability to establish successful pregnancies, even though fertilization may be achieved. Fertil Steril 50:283, 1988 Sperm morphology evaluated by new, strict criteria is a good predictor of in vitro fertilization (IVF) outcome. 1 2 In the Norfolk program, when preovulatory oocytes are inseminated with motile sperm/ml, patients with normal morphology (normal forms >14%) and with concentration and motility within acceptable limits yield a fertilization rate of 89%. However, when the percentage of normal forms is <14%, the fertilization rate decreases to 49.4%. 2 Within this group, a subgroup with a poor prognosis pattern and markedly impaired fertilizing ability can also be identified. 3 The objectives of this study are (1) to determine whether the fertilization rate of preovulatory oo- Received February 19, 1988; revised and accepted April 28, 1988. *Reprint requests: Sergio Oehninger, M.D., Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 825 Fairfax Avenue, Norfolk, Virginia 23507. cytes in patients with severe abnormal morphology can be improved by increasing insemination concentration at the time of IVF and (2) to evaluate the overall reproductive potential of these patients. MATERIALS AND METHODS Two hundred twenty-eight patients (232 cycles) who participated in the Norfolk IVF program during series 25 to 28 (October 1986 to September 1987) were included in this study. Two hundred female partners in these couples had tubal disease or endometriosis (minimal or mild) as the primary cause of infertility. There were 187 male factor patients, 28 with "pure" male factor and 159 with a combined female factor; all had a diagnosis of teratozoospermia. Female and male patients with antisperm antibodies were excluded from this study. To be included in the study, male patients had to have a sperm concentration >20 X 10 6 /ml and a Oehninger et al. Abnormal sperm morphology in IVF 283
progressive motility of >30% in at least two preliminary basic semen analyses and also at the time of IVF. The purpose of these requirements was to minimize the impact of these two variables on the fertilization rate. 2 3 The 228 men were divided into three groups according to criteria previously established in the Norfolk andrology laboratory: 2 3 (1) normal sperm morphology (>14% normal sperm forms), 41 cases; (2) patients with a "good" prognosis pattern (normal forms between 4% and 14%), 144 cases; and (3) patients with a "poor" prognosis pattern ( <4% normal sperm forms), 43 cases. Sperm morphology was evaluated on the specimen delivered on the day of oocyte retrieval (performed by laparoscopy or transvaginal ultrasound-guided technique). The semen slides were stained by the Diff-Quick technique (Diff-Quick AHS del Caribe, Inc., Aguada, Puerto Rico), 4 and sperm morphology was evaluated by the strict criteria described by Kruger et au- 3 This method of evaluation has an intertechnician and intratechnician variability that is not significant. 3 5 Sperm concentration and percentage of normal motility were assessed by computer analysis ( Cellsoft Semen Analysis System, Cryo Resources Ltd., New York, NY). The IVF procedures used for ovarian stimulation (a combination of two follicle-stimulating hormone, [FSH] /two human menopausal gonadotropin, [hmg]/human chorionic gonadotropin, [hcg] or four FSH/hCG), sperm preparation, oocyte classification, insemination, and culture in the Norfolk program have been described previously. 6 7 In the Norfolk experience all of these protocols have demonstrated provision of preovulatory oocytes with identical fertilization rates. 6 Only mature oocytes (metaphase II at aspiration or metaphase I oocytes which have extruded their polar body) were used in this study. Routinely, motile sperm/mlloocyte were used for oocyte insemination in 3.0 ml of medium. Forty-one patients/cycles from group 1, 74 patients/cycles from group 2, and 15 patients/ cycles from group 3 followed this protocol for insemination. After April 1987, the insemination concentration of patients with <14% normal sperm forms (abnormal morphology) was increased 2- to 10-fold, 100,000 to sperm/mlloocyte. Seventy patients/cycles from group 2 and 32 patients/ cycles from 3 followed this protocol. In the vast majority of these cases (75% of patients from group 2 and 73% of patients from group 3), the insemination concentration was motile sperm/ml/ oocyte. In the rest of the patients and within both groups, the concentration was increased from 2- to 10-fold, thus avoiding any bias toward using different concentrations in the two populations. Throughout the study period, only four couples from group 3 participated in the IVF program twice and thus were subjected to both insemination concentration protocols. Statistical analysis was performed with the ANOV A with the LSD range test (least significant differences) for comparisons among the three groups. The effect of insemination concentration on fertilization and pregnancy rates within each group (routine vs high insemination concentration) was further tested using the chi square test. Values of P < 0.05 were considered significant. Results are presented as mean values ± standard deviation. RESULTS Three groups of patients are presented: group 1, 41 patients, mean number of normal sperm forms 18.4 ± 3.5%; group 2, 144 patients, normal forms 7.3 ± 2.8%; group 3, 43 patients, normal forms 1.4 ± 1.2% (P < 0.05). The spermatozoa abnormalities observed in all cases corresponded to head defects (and a minority of midpiece alterations) as defined previously in the good and poor prognosis pattern groups. 3 The mean number of preovulatory oocytes retrieved per patient (group 1, 3.8 ± 1.8; group 2, 4.0 ± 3.8; group 3, 3.2 ± 1.8) was not significantly different for these groups. Neither were there any differences in the number of oocytes obtained from patients inseminated with sperm/mlloocyte compared with >100,000 sperm/mlloocyte in groups 2 and 3. The number of cycles (IVF attempts), cycles with transfer, ongoing pregnancies, and number of miscarriages for groups 1 to 3 and for the two subgroups within groups 2 and 3 (routine and high insemination concentrations) are presented in Table 1. Overall Results The overall results of all patients in groups 1 to 3 are depicted in Table 2. For group 1 the overall fertilization rate was 94.3%; the per cycle and per transfer pregnancy rates were 43.9%. The miscarriage rate (preclinical and clinical) 8 was 29.7%; the per cycle and per 284 Oehninger et al. Abnormal sperm morphology in IVF Fertility and Sterility
Table 1 Number of Cycles, Cyles with Transfer, Ongoing Pregnancies, and Miscarriages in the Different Groups and Subgroups Group 2 Group 3 Sperm cone. at insem. Sperm cone. at insem. Group 1 Patients >loo,oooa Patients >100,000a No. cycles 41 144 74 70 47 15 32 No. transfers 41 110 54 56 29 5 24 No. ongoing pregnancies 13 28 14 14 2 1 1 No. miscarriages 5 20 12 8 2 2 Total pregnancies 18 48 26 22 4 1 3 a and >100,000 sperm/ml/oocyte utilized for insemination at IVF. transfer ongoing pregnancy rates (>20 weeks) were 31.7%. For group 2 the overall fertilization rate was 85.7%; the per cycle and per transfer pregnancy rates were 33.3% and 43.6%, respectively. The miscarriage rate was 41.6%, and the per cycle and per transfer ongoing pregnancy rates were 19.4% and 25.4%, respectively. For group 3 the overall fertilization rate was 44.5% (P < 0.0001 compared with groups 1 and 2); the pregnancy rate per cycle was 8.5% (P < 0.0003 compared with group 1 and P < 0.01 compared with group 2); the pregnancy rate per transfer was 13.7% (P < 0.003 compared with groups 1 and 2). The miscarriage rate was 50.0%, whereas the ongoing pregnancy rate per cycle was 4.2% (P < 0.001 compared with group 1 and P < 0.05 compared with group 2); the ongoing pregnancy rate per transfer was 6.8% (P < 0.006 compared with group 1 and P < 0.04 compared with group 2). Insemination with versus >100,000 Motile Sperm/ml/Oocyte With sperm/ml/oocyte, the fertilization rate was significantly higher in group 2 than in group 3 (87.8% versus 14.5%, P < 0.0001). With >100,000 sperm/ml/oocyte, there was also a significant difference between groups 2 and 3 (83.9% versus 62.6%, P < 0.03), although the difference was clearly less pronounced. For group 2 the fertilization rate, pregnancy rate, and ongoing pregnancy rate were similar with both insemination protocols (Table 2). The fertilization rate (87.8% versus 83.9%), pregnancy rate per cycle (35.1% versus 31.4%) and per transfer (48.1% versus 39.2% ), miscarriage rate (46.1% versus 36.3%), and ongoing pregnancy rate per cycle (18.9% versus 20.0%) and per transfer (25.4% versus 17.8%) were not significantly different with the routine and high sperm concentrations for insemination. Table 2 Fertilization Rates and Pregnancy Outcome in the Different Groups and Subgroups Group 1 n = 41 cycles Group 2 Group 3 n = 74 >100,000 >100,000 n = 70 cycles n = 15 n = 32 Fertilization rate 94.3 Pregnancy rate/cycle 43.9 Pregnancy rate/transfer 43.9 Miscarriage rate 29.7 Ongoing pregnancy rate/cycle 31.7 Ongoing pregnancy rate/transfer 31.7 85.7 33.3 43.6 41.6 19.4 25.4 87.8 35.1 48.1 46.1 18.9 25.9 83.9 44.5a 14.5b 62.6b 31.4 8.5' 6.6 9.3 39.2 13.7d 20.0 12.5 36.3 50.0 66.6 20.0 4.2 6.6 3.1 17.8 6.8' 20.0 4.1 results are expressed in%. a P < 0.0001 compared with groups 1 and 2. b p < 0.005. 'P < 0.0003 compared with group 1; P < 0.01 compared with d P < 0.003 compared with groups 1 and 2. P < 0.001 compared with group 1; P < 0.05 compared with t P < 0.006 compared with group 1; P < 0.04 compared with Oehninger et al. Abnormal sperm morphology in IVF 285
For group 3 the fertilization rate with sperm/ml/oocyte was 14.5%, compared with 62.6% with >100,000 sperm/ml/oocyte (P < 0.005). The pregnancy rate per cycle (6.6% versus 9.3%) and per transfer (20.0% versus 12.5%) and the ongoing pregnancy rate per cycle (6.6% versus 3.1%) and per transfer (20.0% versus 4.1%) were not significantly different among the patients subjected to the routine and the high sperm concentrations for insemination. In the group of patients in whom >100,000 sperm/mlloocyte were used, the miscarriage rate was 66.6%, not significantly different from the miscarriage rate of groups 1 and 2. Table 3 presents the results of the four couples who were subjected to the routine and increased insemination concentrations in two consecutive IVF attempts. The incidence of polyspermia was approximately 8.0% (preovulatory oocytes) in all groups, similar to results previously reported by the Norfolk program.9 DISCUSSION Semen analysis continues to be the most important tool for the evaluation of male fertility potential.10 The assessment of sperm morphology, one of the three most important parameters-concentration and motility being the other two-needs to be improved considerably from the routine standards of evaluation. Early evidence suggested that when this parameter is evaluated by strict criteria, it is more valuable than any other traditional semen characteristics as a prognosticator of sperm fertilizing capacity in IVF. 1 11 For this reason, we now use the new strict criteria described by Kruger et al. 1-3 for the diagnosis of sperm morphology. The identification of patients with good or poor prognosis patterns 3 gives the clinician valuable predictive information as long as oocyte quality, maturation stage diagnosis, insemination, and culture are performed under well-controlled laboratory conditions.7 Furthermore, a more accurate diagnosis of sperm abnormalities may explain cases of otherwise unexplained failure of fertilization in IVF. 12 In this study, we have examined the effect of a single parameter-insemination concentrationon IVF results in patients with abnormal morphology (<14% normal forms) while maintaining the constancy of other sperm variables-concentration and motility. Consequently, in terms of semen and oocyte factors, the groups proved to be well matched. The use of strict criteria allows the identification of a group of patients with severe sperm head abnormalities who have a poor prognosis in IVF with the routine insemination concentration of motile sperm/mlloocyte. 2 In this study, patients with <4% normal forms and with concentration and motility within acceptable limits (pure teratozoospermia) had a fertilization rate of 14.5% with the above mentioned insemination concentration, confirming our previous results. 3 In an effort to increase the fertilization rate in patients with severely abnormal sperm forms, we increased the insemination concentration 2- to 10-fold. No differences were observed in those patients with a good prognosis pattern (4% to 14% normal sperm forms); however, the fertilization rate in the poor prognosis pattern group ( <4% normal forms) was significantly higher (14.5% compared with 62.6%) when the sperm concentration was increased (100,000 to motile sperm/ml/oocyte) at the time of IVF. Wolf et ap 3 found the highest fertilization rates in oligospermic men with motile sperm/mi. Among patients with abnormal concentration, motility, or morphology, Diamond et al. 14 also reported that insemination with up to Table 3 Results of Four Couples That Were Subjected to Both Insemination Concentrations ( of Them Belonged to the Category of Poor Prognosis) Insemination Concentration (sperm/ml/oocyte) No. Preovulatoy/Oocytes Fertilized/Inseminated Outcome Patient 1 Patient 2 Patient 3 Patient 4 0/4 4/4 0/2 0/3 3/4 2/2 2/5 1/2 No transfer No transfer Pregnant (clinical miscarriage) Pregnant (ongoing) 286 Oehninger et al. Abnormal sperm morphology in IVF Fertility and Sterility
motile sperm/ml/oocyte resulted in an increased fertilization rate in IVF. In addition, the use of a higher number of motile sperm in GIFT procedures was recently found to be an effective therapy in the treatment of oligospermic infertility.15 Although fertilization was achieved in the group with a poor prognosis pattern, pregnancy outcome did not improve. The pregnancy rates per cycle and per transfer remained very low, and the ongoing pregnancy rates per cycle (3.1%) and per transfer (4.2%) were still extremely poor. The miscarriage rate in patients with abnormal morphology (groups 2 and 3) was higher than in the patients with normal sperm forms, although this difference did not attain statistical significance. However, the trend suggests that the sperm head defects may be markers for other sperm defects that significantly impair fertility. Sperm nucleus defects have been associated with infertility, 10 and the association of teratozoospermia, chromosomal aberrations, and male infertility has been documented.10 16 It is possible that abnormal sperm head morphology reflects an abnormality in spermatogenesis that is manifested by decreased fertilizing capacity17 and the genesis of embryos with a low potential for establishing a normal pregnancy. Are some of these patients at a higher risk for poor embryo development and miscarriage after natural fertilization? Preliminary data from our clinical program after analysis of the reproductive history of these patients suggest that the spontaneous term pregnancy rate is very low, whereas the miscarriage rate is higher than in patients with normal morphology. Patients with severe sperm head abnormalities have a lower ability to establish successful pregnancies following IVF. Even though fertilization may be achieved by corrective measures, their overall reproductive potential seems to be poorer. Acknowledgments. The authors gratefully acknowledge the contributions of Ms. Debbie Jones, statistician; Charlotte Schrader, Ph.D., editor; and Mrs. Helen Ashberry, secretary. REFERENCES 1. Kruger TF, Menkveld R, Stander FSH, Lombard CJ, Van der Merwe JP, van Zyl JA, Smith K: Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril46:1118, 1986 2. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Veeck LL, Morshedi M, Brugo S: A new method of evaluating sperm morphology with predictive value for in vitro fertilization. Urology 30:248, 1987 3. Kruger T, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S: Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 49:112, 1988 4. Kruger TF, Ackerman SB, Simmons KF, Swanson RJ, Brugo S, Acosta AA: A quick reliable staining technique for sperm morphology. Arch Androl 18:275, 1987 5. Menkveld R: The influence of environmental factors on spermatogenesis and sperm parameters. Doctoral thesis: University of Stellenbosch, Parow, South Africa, 1987 6. Acosta AA, Bernardus RE, Jones GS, Garcia J, Rosenwaks Z, Simonetti S, Veeck LL, Jones D: The use of pure FSH alone or in combination for ovulation stimulation in in vitro fertilization. Acta Eur Fertil 16:81, 1985 7. Veeck LL, Maloney M: Insemination and fertilization. In In Vitro Fertilization-Norfolk, Edited by HW Jones Jr, GS Jones, GD Hodgen, Z Rosenwaks. Baltimore, Williams & Wilkins, 1986, p 168 8. Jones HW Jr, Acosta AA, Andrews MC, Garcia JE, Jones GS, Mantzavinos T, McDowell J, Sandow BA, Veeck L, Whibley TW, Wilkes CA, Wright GL Jr: What is a pregnancy?: a question for programs of in vitro fertilization. Fertil Steril 40:728, 1983 9. 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Diamond MP, Rogers BJ, Vaughn WK, Wentz AC: Effect of the number of inseminating sperm and the follicular stimulation protocol on in vitro fertilization of human oocytes in male factor and non-male factor couples. Fertil Steril 44:499, 1985 15. Matson PL, Blackledge DG, Richardson PA, Turner SR, Y ovich JM, Y ovich JL: The role of gamete intrafallopian transfer (GIFT) in the treatment of oligospermic infertility. Fertil Steril 48:608, 1987 16. Abramsson L, Beckman G, Duchek M, Nordenson I: Chromosomal aberrations and male infertility. J Urol 128:52, 1982 17. Marsh SK, Bolton VN, Brande PR: The effect of morphology on the ability of human spermatozoa to penetrate zonafree hamster oocytes. Human Reprod 2:499, 1987 Oehninger et al. Abnormal sperm morphology in IVF 287