An evaluation of various treatments to increase sperm penetration capacity for potential use in an in vitro fertilization program

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1 FERTILITY AND STERILITY Copyright c 1992 The American Fertility Society Printed on ocid-free paper in U.S.A. An evaluation of various treatments to increase sperm penetration capacity for potential use in an in vitro fertilization program Douglas T. Carrell, M.S.*t WilliamS. Bradshaw, Ph.D.* Kirtly P. Jones, M.D.~ Richard G. Middleton, M.D.t C. Matthew Peterson, M.D.~ Ronald L. Urry, Ph.D.U Brigham Young University, Provo, and University of Utah School of Medicine, Salt Lake City, Utah Objective: To select in vitro fertilization (IVF) patients with a low sperm penetration assay (SPA) value and to determine if the penetration rate, fertilization rate, and the pregnancy rate (PR) can be improved in these patients by treating sperm with refrigeration, calcium ionophore A23187, prostaglandin E 1, prostaglandin E 2, or heparin. Design: The study consists of three parts: identification of patients with poor SPA values, analysis of treatments to improve the SPA value, and evaluation of the treatments to improve fertilization andprs. Results: The data indicate that treatment of sperm with refrigeration can improve fertilization and PRs during IVF in selected patients previously shown to have an improved SPA value with the treatment. Conclusions: Three points are emphasized: (1) the treatments analyzed in this study can improve SPA values in some of the patients with low sperm penetration capacity; (2) of the treatments studied, sperm refrigeration resulted in the largest improvement in sperm penetration capacity; and (3) sperm refrigeration can increase fertilization and PRs during IVF in this select group of patients. Fertil Steril1992;57:134-8 Sperm capacitation involves necessary biochemical and morphological changes that are part of the fertilization process. Although the exact mechanism of the capacitation process remains to be clarified, capacitation has been shown to be associated with an influx of calcium ions and an effiux of hydrogen ions through the sperm plasma membrane, along with an effiux of cholesterol and a decrease in the net negative charge of the plasma membrane. 1 The sperm penetration assay (SPA) is considered a useful tool in diagnosing impairments in the capacitation process by evaluating the ability of sperm to pene- Received March 11, 1991; revised and accepted September 11, *Department of Zoology, Brigham Young University. t Division of Urology, University of Utah School of Medicine. :j: Department of Obstetrics and Gynecology, University of Utah School of Medicine. Reprint requests: Ronald L. Urry, Ph.D., Division of Urology, University of Utah School of Medicine, Salt Lake City, Utah trate zona-free hamster ova. 2-4 Although some controversy remains, SPA results have generally been shown to correlate with the ability to fertilize human ova during in vitro fertilization (IVF) Several substances have been used to stimulate capacitation in various mammalian species by increasing calcium transport or by altering other capacitation related events. Aitken et al.u demonstrated that sperm from known fertile donors incubated with calcium ionophore A23187 had a significant increase in the ability to penetrate zonafree hamster ova. Valencia et al. 12 reported that sperm not allowed sufficient incubation time to undergo capacitation are unable to penetrate zona-free hamster ova. Penetration did occur if heparin was added to the culture medium. 12 Prostaglandin E 1 and PGE2, which increase adenylate cyclase activity and are also believed to act as calcium ionophores in sperm, have been shown to increase sperm penetration capacity _IS Bolanos et aly reported that sperm stored at 2 to 5 C in a zwitterionic, yolk-supple- 134 Carrell et al. Enhancement of sperm penetration

2 mented (TEST -yolk) buffer also may have increased penetration capacity. Preliminary data previously reported by us demonstrated that the SPA may be useful to identify a subpopulation of patients with decreased SPA values that can be improved by preincubating the sperm with a capacitation inducer before their addition to ova. 15 Subsequently, others have investigated the usefulness of refrigeration in TEST -yolk buffer to improve sperm penetration and reported varying degrees of success The purpose of this study was to select patients, before IVF, with decreased SPA values and to determine if penetration rates could be increased by sperm refrigeration in TESTyolk buffer or incubation with heparin, calcium ionophore A23187, PGE 1, or PGE 2, then evaluate the effectiveness of the selected technique to improve fertilization during IVF. MATERIALS AND METHODS Sperm Penetration Assay Semen samples were obtained from 4 76 patients no >90 days before IVF. After liquefaction, approximately 1.0 ml of the sample was used for a standard semen analysis, 20 and the remaining portion of the sample was used for the SPA. The SPA was performed as previously described 21 except for the following changes. The medium used throughout the procedure was modified Ham's F -10 medium (GIDCO, Grand Island, NY) supplemented with 8 heatdeactivated human serum. After two washes, sperm were incubated in a humidified, 37 C, 5 C0 2 incubator overnight. The sperm were then adjusted to a concentration of 5.0 X 10 6 progressively motile sperm/ml. Approximately 0. 7 ml of the sperm suspension was placed in the center well of a 50 X 60-mm center well Petri dish (Falcon Plastics, Oxnard, CA) to which 30 to 40 zona-free hamster ova were added. Repeat Sperm Penetration Assay In 56 patients whose initial SPA value was <10 penetration, a modified SPA procedure was used to evaluate a second sample. A portion of the fresh sample was mixed 1:1 with TEST-yolk buffer (chemicals from Sigma, St. Louis, MO and :o;;1-dayold filtered egg yolk obtained locally) and incubated overnight at 4 C as described by Bolanos et al. 14 The solution was then warmed to 37 C, washed two times in modified Ham'S F-10 medium, adjusted to a concentration of 5.0 X 10 6 motile sperm/ml, and incubated for 2 hours before use in the SPA. The remaining portion of the semen sample was washed two times in medium and incubated at 37 C overnight. The sperm sample was then divided into aliquots that were incubated for 45 minutes with medium containing one of the following chemicals: 75 #LM calcium ionophore A23187 (Sigma), 50 #LM PGE 1 (Sigma), 50 #LM PGE 2 (Sigma), and 50 units (USP/mL) of heparin (Wyeth, Philadelphia, PA). An additional control aliquot without chemical treatment underwent a serum swim-up procedure. 20 The samples were then washed and resuspended at a concentration of 5.0 X 10 6 motile sperm/ml and incubated at 37 C for 2 hours before the addition of zona-free oocytes. In Vitro Fertilization In six patients who had initial and repeat SPA values of 0 penetration with the serum swim-up treated sperm, and >20 penetration with refrigeration-treated sperm, a modified IVF procedure was used. Oocytes recovered during IVF from these patients were divided into two groups with equal numbers of morphological normal oocytes (normal amount and distribution of cumulus with polar body) per group. One group of oocytes was incubated with sperm prepared by serum swim-up, whereas the other group was incubated with sperm prepared by the refrigeration technique described above. One hundred forty-three additional patients with SPA values < 10 were also evaluated for the effectiveness of sperm refrigeration in improving IVF. Sixty-two of the patients underwent IVF using only a fresh semen sample prepared by serum swim-up. Eighty-one of the patients underwent IVF using a semen sample that was refrigerated overnight, which was then mixed with another fresh sample prepared by serum swim-up. The number of progressively motile sperm added to each ovum varied from 1.0 X 10 5 to 6.0 X 10 5 in both treatment groups depending on previous sperm quality (poorer initial quality had higher sperm concentrations). Acrosome Reaction Fresh sperm prepared by serum swim-up before IVF were compared with those prepared by refrigeration to determine the percentage of acrosomereacted sperm. An aliquot of the sperm suspension was removed and analyzed by the triple-stain procedure 22 both immediately after the sperm preparation and again 18 hours later. A second aliquot Carrell et al. Enhancement of sperm penetration 135

3 Table 1 Sperm Penetration Values After Various Sperm s in Patients With an Initial Penetration Capacity < 10 (n:56) Serum swim-up Refrigeration Heparin Ionophore A23187 PGE 1 PGE2 Penetration 7.2 ± ± 4.6b,c 11.8 ± 1.6d 13.2 ± 2.1d 13.4 ± 2.3d 5.9 ± 2.4 Patients improving to >20 penetration b P < 0.01 compared with serum swim-up. c P < 0.05 compared with all other treatment groups. d P < 0.05 compared with serum swim-up was removed at 18 hours and incubated with A23187 for 30 minutes then analyzed by the triple-stain procedure. Statistics The data were analyzed by ANOV A. Log transformations were performed on penetration rates for distribution normalization. RESULTS Table 1 shows the sperm penetration values after the five sperm treatments from patients initially penetrating <10 of the ova. Penetration values were significantly improved by treatment with refrigeration, heparin, ionophore A23187, and PGE 1 Seventy-seven percent of patients with initial values < 10 improved to >20 after refrigeration. In vitro fertilization rates were significantly (P < 0.05) higher for ova inseminated with refrigeration prepared sperm in five of the six IVF patients with 0 SPA values, compared with those inseminated by sperm prepared with serum swim-up samples. The mean (±SE) percent fertilization for the refrigerated samples was 51.2 ± 12.5 compared with 25.0 ± 16.8 for the serum swim-up samples. Live births resulted in two of the patients who had fertilization only from the refrigeration preparation. Table 2 compares semen quality for the 143 patients studied in the two sperm preparation groups during IVF. Initial semen quality was similar for both groups, although the percent of progressively motile sperm was significantly decreased in the group undergoing IVF with both serum swim-up and refrigerated sperm compared with serum swim-up only (P < 0.05). The mean age of the wife undergoing IVF was 32 ± 0.9 years for the refrigeration group and 30.8 ± 0.8 for the serum swim-up group. The mean fertilization rate (Table 3) was significantly increased in the refrigeration group ( 66.2 ± 2.9) compared with the serum swim-up group (47.1 ± 3.9). The pregnancy rate (PR) was 26 for the refrigeration group compared with 11.3 for the serum swim-up group. Data comparing the percentage of viable, acrosome-reacted sperm from both groups is shown in Table 4. The percentage of acrosome-reacted sperm was significantly increased in the refrigeration group. Both groups had an increase in the percentage of acrosome-reacted sperm after incubation with calcium ionophore A DISCUSSION Despite the fact that the SPA has been used for the last 13 to 14 years, some controversy remains as to the usefulness of this assay in predicting fertilization rates in patients undergoing IVF At our laboratory, a hamster egg penetration value of <10 has correlated with a lower fertilization rate compared with patients with a penetration value of > 15. Because of this, we undertook this series of studies to determine if various types of sperm manipulation could improve the sperm penetration capacity and whether this would improve IVF rates in this select group of patients. Table 2 Comparison of Initial Semen Quality for 143 IVF Patients Studied With Two Different Sperm Penetrations I. Serum swim-up refrigeration Initial No. of SPA patients penetrated ± 0.6" ± 0.8 Morphology oval 61.6 ± ± 1.4 Reacted Count (total Hypo- Progressive progressively osmolarity motility Count motile) Xl0 6 /cc xw 63.8 ± ± 1.8b ± 15.1 b ± 14.3b 58.3 ± ± ± ± 12.4 b P < 0.05 compared with treatment group. 136 Carrell et al. Enhancement of sperm penetratwn

4 Table 3 Comparison of Fertilization and PRs for 143 IVF Patients With Sperm Prepared by Refrigeration or by Refrigeration Plus Serum Swim-up No. of Patients Ongoing patients Fertilization < 33 fertilization pregnancy I. Serum swim-up ± 3.9" refrigeration ± 2.9b Values are percent means ± SE. As demonstrated in this paper, sperm penetration capacity into zona-free hamster eggs can be increased with sperm refrigeration, incubation of sperm with heparin, calcium ionophore, and PGE 1 Approximately 75 of all patients with hamster egg penetration values of <10 improve to >20 after sperm refrigeration. Approximately,25 of patients improved with heparin to values > 20 when the initial penetration value was <10. with the ionophore and PGE 1 also contributed to approximately 30 ofthe patients increasing to >20 penetration. Prostaglandin E 2 had no effects on sperm penetration capacity. Using the above information, a small subgroup of patients was identified that had repeated hamster egg penetration values ofo. Patients were selected for this group if they had initial values of 0 that could be improved to >20 with refrigeration. This group of patients subsequently underwent IVF, and it is interesting to note that four of the six patients had no fertilization in the portion of oocytes that had sperm added from conventional sperm preparation techniques. All of these four patients had at least some fertilization with refrigerated prepared sperm. Although the number of patients with zero penetration values is small, it does suggest that there is a group of select patients that may benefit from refrigeration and/or heparin incubation before adb P < 0.05 compared with serum swim-up group. dition of the sperm to the oocytes. More importantly, a larger population of patients with initial hamster egg penetration values of <10 but >0 underwent IVF and had either sperm prepared by conventional techniques added to the oocytes or had refrigerated prepared sperm combined with sperm prepared through serum swim-up techniques added to the oocytes. The group of patients that had refrigerated plus serum swim-up prepared sperm had higher fertilization rates and had substantially higher PRs compared with patients without the refrigerated prepared sperm. Data from this larger group of patients suggest that for individuals with a hamster egg penetration value of <10, IVF outcome may be improved by combining a refrigerated sample along with the conventionally prepared sample during IVF procedures. This paper further demonstrated that the percent of acrosome-reacted sperm and the percent of acrosome-reacted sperm after an acrosome stimulation test was higher for refrigerated sperm than for serum swim-up prepared sperm. This may be part of, although not the entire explanation, of why the fertilization rate appears to be higher in this group of patients. Additional studies are under way to further explore the mechanisms by which these procedures may enhance fertilization in this select group of individuals. It will also be necessary to study additional Table 4 Comparison of Acrosome-Reacted Sperm Before and After With Ionophore A23187 in the Two Groups Postsperm preparation viable, acrosome-reacted 18 h postsperm preparation viable, acrosome-reacted Original sample A23187 treatment Original sample A23187 treatment I. Serum swim-up refrigeration 12.4 ± o.8" 18.4 ± ± ± 2.6b 20.0 ± ± ± 2.0b 41.4 ± 3.2b b P < 0.05 compared with serum swim-up group. Carrell et al. Enhancement of sperm penetration 137

5 numbers of patients to determine the exact role, if any, of these procedures in benefiting couples undergoing IVF. It is extremely important to re-emphasize that this group of individuals is a highly select group that was first shown to have a low sperm penetration value that could be improved by specific sperm manipulation procedures. Although we have not found any deleterious effect using this sperm preparation procedure, neither have we suggested that an improvement in fertilization rates will be found in individuals with a normal initial hamster egg penetration value. REFERENCES 1. Langlais J, Roberts KD: A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian sperm. Gamete Res 12:183, Paulsen CA: Another look at the sperm penetration assay. Fertil Steril 40:302, Rogers BJ: The sperm penetration assay: its usefulness reevaluated. Fertil Steril 43:821, Gould JE, Overstreet JW, Yanagimachi H, Yanagimachi R, Katz DF, Hanson FW: What functions of the sperm cell are measured by in vitro fertilization of zona-free hamster eggs? Fertil Steril 40:344, WolfDP, Sokoloski JE, Quigley MM: Correlation of human in vitro fertilization with the hamster egg bioassay. Fertil Steril 40:53, Margalioth EJ, Navot D, Laufer N, Yosef SM, Rabinowitz R, Yarkoni S, Schenker JG: Zona-free hamster ovum penetration assay as a screening procedure for in vitro fertilization. Fertil Steril 40:386, Foreman R, Cohen J, Fehilly CB, Fishel SB, Edward RG: The application of the zona free hamster egg test for the prognosis of human in vitro fertilization. J Vitro Fert Embryo Transfer 1:166, Mao C, Grimes DA: The sperm penetration assay: can it be discriminate between fertile and infertile men? Am J Obstet Gynecol 159:279, Rogers BJ: The sperm penetration assay: its usefulness reevaluated. Fertil Steril 43:821, Margalioth EJ, Feinmesser M, Navot D, Mordel N, Bronson RA: The long-term predictive value of the zona-free hamster ova sperm penetration assay. Fertil Steril 52:490, 1989 ll. Aitken RJ, Ross A, Hargreave T, Richardson D, Best F: Analysis of human sperm function following exposure to ionophore A J Androl 5:321, Valencia A, Wens MA, Merchant H, Reyes R, Delgado NM: Capacitation of human sperm by heparin. Arch Androl 12: 109, Aitken RJ: In vitro fertilization for male infertility. Acta Eur Fertil 15:425, Bolanos JR, Overstreet JW, Katz DF: Human sperm penetration of zona-free hamster eggs after storage of the semen for 48 hours at 2 C to 5 C. Fertil Steril 39:536, Urry RL, Carrell DT, Middleton RG: Individualization of sperm capacitation prior to use for insemination, GIFT, or IVF. (Abstr. 123) Presented at the 44th Annual Meeting of The American Fertility Society, Atlanta, Georgia, October 10 to 13, Published by The American Fertility Society, in the Program Supplement, 1988, p Katayama KP, Stehlik E, Roesler M, Jeyendran RS, Holmgren WJ, Zaneveld LJD: of human spermatozoa with an egg yolk medium can enhance the outcome of in vitro fertilization. Fertil Steril52:1077, Rogers BJ: Use of test-yolk buffer in human sperm processing for in vitro fertilization (IVF). (Abstr. 32) Presented at the 15th Annual Meeting of the American Society of Andrology, Columbia, South Carolina, April 6 to 9, Published by the American Society of Andrology, in the Program Supplement, 1990, p Paulson RJ, Sauer MV, Francis MM, Macaso TM, Lobo RA: Use of test-yolk buffer (TYB) to enhance fertilization during human in vitro fertilization (IVF) in cases of suspected male infertility. (Abstr. P-16) Presented at the 38th Annual Meeting of the Pacific Coast Fertility Society, Scottsdale, Arizona, April 25 to 29, Published by the Pacific Coast Fertility Society, in the Program Supplement, 1990, p All 19. Rogers BJ: The use of test-yolk buffer in intrauterine inseminations. (Abstr. P-15) Presented at the 38th Annual Meeting of the Pacific Coast Fertility Society, Scottsdale, Arizona, April 25 to 29, Published by the Pacific Coast Fertility Society, in the Program Supplement, 1990, pall 20. Urry RL: Laboratory diagnosis of male infertility. In Clinics in Laboratory Medicine, Vol. 5, Edited by GB Schumann. Philadelphia, W.B. Saunders Company, 1985, p Urry RL, Carrell DT, Hull DB, Middleton RG, Wiltbank MC: Penetration of zona-free hamster ova and bovine cervical mucus by fresh and frozen human spermatozoa. Fertil Steril 39:690, Talbot P, Chacon RS: A triple-stain technique for evaluating normal acrosome reactions of human sperm. J Exp Zool215: 201, Foreman R, Cohen J, Fehilly CB, Fishel SB, Edwards RG: The application of the zona-free hamster egg test for the prognosis of human in vitro fertilization. J In Vitro Fert Embryo Transfer 1:166, Carrell et al. Enhancement of sperm penetration