Vol. 58, No.6, December 1992
FERTILITY AND STERILITY Copyright
©
Printed on acid-free paper in U.S.A.
1992 The American Fertility Society
Enhanced gamete interaction in the sperm penetration assay after coincubation with pentoxifylline and human follicular fluid*
Hovey Lambert, Ph.D.t Alex Steinleitner, M.D.:\: Juergen Eisermann, M.D.
Nurys Serpa, B.S. Bernard Cantor, M.D.
Department of Obstetrics and Gynecology, Mt. Sinai Medical Center, Miami Beach, Florida
Objective: To evaluate the effect of pentoxifylline and heat-inactivated human follicular fluid (FF) on performance in the sperm penetration assay (SPA) as a paradigm for the effect of these agents on human sperm-egg interaction in vivo and in vitro fertilization. Design: Semen specimens from men undergoing SPA testing for evaluation of suspected male factor infertility were coincubated with neat medium or media supplemented with pentoxifylline or human FF in a nonblinded manner. Participants: Twenty male factor infertility patients. Interventions: Semen specimens were preincubated with: [1] pentoxifylline 0.25 mg/mL; [2] 10% human FF; [3] pentoxifylline + human FF; and [4] neat Biggers, Whitten, and Whittingham medium. Main Outcome Measures: Differences in the rate of penetration of zona-free hamster oocytes. Results: Preincubation with either human FF or pentoxifylline produced a significant improvement in hamster egg penetration rates. Coincubation with a combination of human FF and pentoxifylline resulted in a significant enhancement of penetration as compared with single agent treatment. Conclusions: Coincubation of sperm with human FF and pentoxifylline may provide a means of enhancing sperm activity for insemination and assisted reproduction. Fertil Steril 1992;58:1205-8 Key Words: Human follicular fluid, pentoxifylline, male factor infertility
Treatment of male factor infertility remains one of the most problematic aspects of clinical reproductive medicine. Although the precise pathophysiology underlying sperm dysfunction is not defined in most individuals, in general terms patients may be classified as suffering from abnormalities of sperm locomotion and energy source utilization or abnormalities of capacitation and cell-cell recognition. Received January 28, 1991; revised and accepted August 13, 1992. * Presented at the 46th Annual Meeting of The American Fertility Society, Washington, DC, October 15 to 19,1990. t Present address: San Francisco Center For Reproductive Medicine, San Francisco, California. :j: Reprint requests and present address: Alex Steinleitner, M.D., San Francisco Center For Reproductive Medicine, 390 Laurel Street, Suite 100, San Francisco, California 94118. Vol. 58, No.6, December 1992
Traditional therapeutic modalities have emphasized nonspecific treatments such as sperm washing and intrauterine insemination (lUI). More recently, investigators have reported the use of agents such as egg yolk buffer solutions, human follicular fluid (FF), maternal blood serum, and pentoxifylline to enhance the outcome of lUI and in vitro fertilization (IVF) (1-3). Most recent reports (1-3) have described the use of a single additive for the treatment of sperm dysfunction. Because many male factor patients appear to suffer from abnormalities of multiple aspects of sperm function, the use of multimodality therapy to enhance both locomotion and capacitation seems intuitively promising. In this report, we describe the effect of preincubation with a combination of the phosphodiesterase inhibitor pentoxifylline and hu-
Lambert et al.
Enhanced SPA with pentoxifylline and human FF
1205
man FF on sperm performance in asthenospermic males as measured by the sperm penetration assay (SPA). MATERIALS AND METHODS Sperm Preparation
Semen specimens were obtained from 20 men who were undergoing evaluation for male factor infertility. Each patient had been previously shown to have abnormalities of sperm motion parameters or morphology on semen analysis. Specimens obtained from donors of proven fertility were employed as controls. Semen samples were allowed to liquefy for 30 minutes at 37°C. Concentration and motility were assessed using a Makler chamber (Sefi-Medical Instruments, Haifa, Israel). The sample was divided into 0.5-mL aliquots and layered with 3.0 mL of Biggers, Whitten, and Whittingham (BWW) medium. The layers of BWW with swim-up sperm populations were incubated overnight in 5% CO 2 at 37°C. Sperm Penetration Assay
The hamster egg penetration assay was performed by the method of Rogers (4). Sexually mature female golden hamsters (6 to 8 weeks old) were subjected to ovarian hyperstimulation with intraperitoneal (lP) injections of 40 IU of pregnant mare's serum gonadotropins (Sigma Chemical Company, St. Louis, MO) on cycle day 1 followed by 40 IU IP of human chorionic gonadotropins (hCG, Sigma Chemical Company) 52 to 56 hours later. Sixteen hours after hCG administration, hamsters were killed by cervical dislocation for oocyte collection. The cumulus cell mass was dissipated with 0.1 % hyaluronidase (Sigma Chemical Company). Zonae pellucidae were removed by treatment with 0.1 % trypsin (Sigma Chemical Company). Eggs were rinsed twice and transferred to wells containing sperm suspensions. Sperm samples were adjusted to a final concentration of 1 X 106 spermatozoa/mL under the following conditions: neat BWW medium, pentoxifylline (0.25 mg/mL, Sigma Chemical Company) in BWW, 10% (vol/vol) human FF in BWW, and pentoxifylline 0.25 mg/mL and 10% human FF in BWW. After a 150-minute incubation, oocytes were mounted and read. Penetration of hamster eggs was determined by the number of swollen sperm heads associated with midpiece and tails in the ooplasm. Patients having penetration rates (percent 1206
Lambert et al.
of hamster eggs penetrated) of
FERTILITY AND STERILITY Copyright
©
Printed on acid-free paper in U.S.A.
1992 The American Fertility Society
Enhanced gamete interaction in the sperm penetration assay after coincubation with pentoxifylline and human follicular fluid*
Hovey Lambert, Ph.D.t Alex Steinleitner, M.D.:\: Juergen Eisermann, M.D.
Nurys Serpa, B.S. Bernard Cantor, M.D.
Department of Obstetrics and Gynecology, Mt. Sinai Medical Center, Miami Beach, Florida
Objective: To evaluate the effect of pentoxifylline and heat-inactivated human follicular fluid (FF) on performance in the sperm penetration assay (SPA) as a paradigm for the effect of these agents on human sperm-egg interaction in vivo and in vitro fertilization. Design: Semen specimens from men undergoing SPA testing for evaluation of suspected male factor infertility were coincubated with neat medium or media supplemented with pentoxifylline or human FF in a nonblinded manner. Participants: Twenty male factor infertility patients. Interventions: Semen specimens were preincubated with: [1] pentoxifylline 0.25 mg/mL; [2] 10% human FF; [3] pentoxifylline + human FF; and [4] neat Biggers, Whitten, and Whittingham medium. Main Outcome Measures: Differences in the rate of penetration of zona-free hamster oocytes. Results: Preincubation with either human FF or pentoxifylline produced a significant improvement in hamster egg penetration rates. Coincubation with a combination of human FF and pentoxifylline resulted in a significant enhancement of penetration as compared with single agent treatment. Conclusions: Coincubation of sperm with human FF and pentoxifylline may provide a means of enhancing sperm activity for insemination and assisted reproduction. Fertil Steril 1992;58:1205-8 Key Words: Human follicular fluid, pentoxifylline, male factor infertility
Treatment of male factor infertility remains one of the most problematic aspects of clinical reproductive medicine. Although the precise pathophysiology underlying sperm dysfunction is not defined in most individuals, in general terms patients may be classified as suffering from abnormalities of sperm locomotion and energy source utilization or abnormalities of capacitation and cell-cell recognition. Received January 28, 1991; revised and accepted August 13, 1992. * Presented at the 46th Annual Meeting of The American Fertility Society, Washington, DC, October 15 to 19,1990. t Present address: San Francisco Center For Reproductive Medicine, San Francisco, California. :j: Reprint requests and present address: Alex Steinleitner, M.D., San Francisco Center For Reproductive Medicine, 390 Laurel Street, Suite 100, San Francisco, California 94118. Vol. 58, No.6, December 1992
Traditional therapeutic modalities have emphasized nonspecific treatments such as sperm washing and intrauterine insemination (lUI). More recently, investigators have reported the use of agents such as egg yolk buffer solutions, human follicular fluid (FF), maternal blood serum, and pentoxifylline to enhance the outcome of lUI and in vitro fertilization (IVF) (1-3). Most recent reports (1-3) have described the use of a single additive for the treatment of sperm dysfunction. Because many male factor patients appear to suffer from abnormalities of multiple aspects of sperm function, the use of multimodality therapy to enhance both locomotion and capacitation seems intuitively promising. In this report, we describe the effect of preincubation with a combination of the phosphodiesterase inhibitor pentoxifylline and hu-
Lambert et al.
Enhanced SPA with pentoxifylline and human FF
1205
man FF on sperm performance in asthenospermic males as measured by the sperm penetration assay (SPA). MATERIALS AND METHODS Sperm Preparation
Semen specimens were obtained from 20 men who were undergoing evaluation for male factor infertility. Each patient had been previously shown to have abnormalities of sperm motion parameters or morphology on semen analysis. Specimens obtained from donors of proven fertility were employed as controls. Semen samples were allowed to liquefy for 30 minutes at 37°C. Concentration and motility were assessed using a Makler chamber (Sefi-Medical Instruments, Haifa, Israel). The sample was divided into 0.5-mL aliquots and layered with 3.0 mL of Biggers, Whitten, and Whittingham (BWW) medium. The layers of BWW with swim-up sperm populations were incubated overnight in 5% CO 2 at 37°C. Sperm Penetration Assay
The hamster egg penetration assay was performed by the method of Rogers (4). Sexually mature female golden hamsters (6 to 8 weeks old) were subjected to ovarian hyperstimulation with intraperitoneal (lP) injections of 40 IU of pregnant mare's serum gonadotropins (Sigma Chemical Company, St. Louis, MO) on cycle day 1 followed by 40 IU IP of human chorionic gonadotropins (hCG, Sigma Chemical Company) 52 to 56 hours later. Sixteen hours after hCG administration, hamsters were killed by cervical dislocation for oocyte collection. The cumulus cell mass was dissipated with 0.1 % hyaluronidase (Sigma Chemical Company). Zonae pellucidae were removed by treatment with 0.1 % trypsin (Sigma Chemical Company). Eggs were rinsed twice and transferred to wells containing sperm suspensions. Sperm samples were adjusted to a final concentration of 1 X 106 spermatozoa/mL under the following conditions: neat BWW medium, pentoxifylline (0.25 mg/mL, Sigma Chemical Company) in BWW, 10% (vol/vol) human FF in BWW, and pentoxifylline 0.25 mg/mL and 10% human FF in BWW. After a 150-minute incubation, oocytes were mounted and read. Penetration of hamster eggs was determined by the number of swollen sperm heads associated with midpiece and tails in the ooplasm. Patients having penetration rates (percent 1206
Lambert et al.
of hamster eggs penetrated) of
