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Peritoneal fluid from patients with endometriosis decreases sperm binding to the zona pellucida in the hemizona assay: a preliminary report*
FERTILITY AND STERILITY
Vol. 57, No.4, April 1992
Copyright" 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Peritoneal fluid from patients with endometriosis decreases sperm binding to the zona pellucida in the hemizona assay: a preliminary report*
Charles C. Coddington, M.D.t:!:§ Sergio Oehninger, M.D.t Dean S. Cunningham, Ph.D., L.C.D.R., M.C., U.S.N.:!:
Keith Hansen, L.C.D.R., M.C., U.S.N.R.:!: Carlos E. Sueldo, M.D. II Gary D. Hodgen, Ph.D.t
The Jones Institute for Reproductive Medicine, Eastern Virginia Medical Schoo~ Norfolk; Portsmouth Naval Hospita~ Portsmouth, Virginia; and Valley Medical Center, University of California, Fresno, California
Objective: To evaluate the effect of peritoneal fluid (PF) from patients with endometriosis on gamete interaction under hemizona assay (HZA) conditions. Design: The HZA was used to study the effect of PF from patients with endometriosis on sperm binding to the zona pellucida using media and normal PF as controls. Setting: The patients were collected from a university hospital infertility clinic. Patients: Peritoneal fluid from 16 women being evaluated for infertility or sterilization who were found to have endometriosis at surgery was used. Three normal patients, who were being sterilized, had PF that was used as a control. Results: Results suggest that there is a significant reduction in the number of tightly bound sperm to the zona surface in endometriosis specimens as reflected in the hemizona index and that this effect is directly related to the stage of the disease. Conclusions: Although the sample is small, this methodology may help to elucidate one of the mechanisms responsible for endometriosis-associated infertility. Fertil SteriI1992;57:783-6 Key Words: Endometriosis, peritoneal fluid, hemizona assay, sperm:oocyte binding, sperm function
Endometriosis is a disease of the female pelvis that is associated with pelvic pain, irregular vaginal bleeding, and infertility. Although mechanical factors can be related to an etiology of infertility with Received July 11, 1991; revised and accepted December 3, 1991.
* Presented in part at the 46th Annual Meeting of The American Fertility Society, Washington, D.C., October 13 to 18, 1990. t The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School. Portsmouth Naval Hospital. § Reprint requests: Charles C. Coddington, M.D., The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia 23507. II Valley Medical Center, University of California.
*
Vol. 57, No.4, April 1992
more severe disease, little is known about the pathophysiology of infertility in the less advanced stages of the disease. Several authors have demonstrated that there are increased numbers of macrophages in the peritoneal cavity of patients with endometriosis (1, 2), but others have questioned this finding (3). Many studies have been performed to evaluate the pelvic cavity environment, but there does not appear to be a clear marker to relate to infertility (1-6). Little information has accumulated about gamete interaction. Studies have been performed to evaluate the effect of peritoneal fluid (PF) on the ability of sperm to penetrate hamster zonafree oocytes (3,4), but the results are conflicting. It has been reported that PF from patients with en-
Coddington et aI.
Endometriotic PF decreased HZA binding
783
dometriosis caused decreased fertility in the mouse model (5) and also caused toxic effects on mouse embryo cleavage (6). Currently, there is not a method that evaluates the effect of PF on the steps of fertilization that occur naturally. The hemizona assay (HZA) has been developed to evaluate tight binding of the sperm to the zona pellucida, a critical event leading to fertilization (7). Studies have confirmed that fertile patients have increased numbers of sperm bound to the zona (7), and increased success in in vitro fertilization has been associated with higher numbers of sperm bound (8). Additionally, sperm bound to the hemizona have undergone the expected acrosome reaction as dem0nstrated by the T-6/antibody with immunofluorescent staining (9). Thus, the HZA is a unique, homologous bioassay by which gamete interaction can be evaluated. Our purpose was to determine the effect of PF from patients with endometriosis on sperm-oocyte interaction under HZA conditions.
washed a second time. The second pellet was overlayed with 0.5 mL of F-10 medium and incubated (37°C, 5% CO2 in air) to achieve a swim-up separation. After 1 hour of incubation, the sperm supernatant was removed and used for testing of hemizona binding. Peritoneal Fluid Peritoneal fluid was obtained from the abdominal cavity of 16 regular cycling women with endometriosis and 3 normal women (controls) who were on days 9 through 15 of their menstrual cycle. Their ages ranged from 25 to 30, and the indication for surgery was for tubal ligation or evaluation of infertility. Endometriosis staging was performed according to current American Fertility Society criteria (10). The fluid was obtained and centrifuged at 200 X g for 10 minutes to remove cellular contents. Supernatant was pipetted into plastic storage tubes and frozen at -4°C. At time of use, the fluid was thawed and warmed before being used in the HZA in a 5:1 dilution with medium containing sperm.
MATERIALS AND METHODS Protocol for HZA Semen Analysis Semen from three fertile donors who had been previously extensively tested in an HZA was used in the studies. The basic semen evaluation was performed using an automated, computerized system (Cell Soft Semen Analyzer, Cryo Resources Ltd., New York, NY). Details of these procedures have been described previously (7). Oocyte Handling and Zona Cutting Human oocytes were obtained from postmortem ovarian tissue. The ovarian tissue was placed on ice after excision, dissected, and minced in phosphatebuffered saline (7). Narishige micromanipulators (Narishige, Tokyo, Japan) mounted on a phase-contrast inverted microscope (Nikon Diaphot, Garden City, NY) were used for equally bisecting the oocytes. The cutting procedures have previously been published (7, 8).
A 100-J'L droplet ofthe sperm suspension (500,000 motile sperm/mL after swim-up) was placed in a Petri dish under oil. In initial experiments, one hemizona was placed in a droplet with sperm that had been exposed to the PF being studied (1:5) (test), whereas the matching hemizona was incubated with a droplet ofsperm exposed to Ham's F-10 and 7.5% fetal cord serum (control). For the second set of experiments, PF from patients with a normal pelvis was used in the control media in the same dilution. The gametes were coincubated for 4 hours (37°C, 5 % CO2 in air). Each hemizona was then removed and rinsed using a fine glass pipette in medium to separate loosely associated sperm (7). The number oftightly bound sperm was counted. Average values were obtained.for number of sperm bound and hemizona index. The hemizona index is the number of sperm bound from test/number of sperm bound from control X 100 (7). A paired t-test was used to compare the hemizona index data.
Sperm Handling for the HZA An aliquot of semen (0.5 mL) was diluted with 1 mL of Ham's F-10 culture medium (GIBCO, Grand Island NY), supplemented with 7.5% heat-inactivated human fetal cord serum. After centrifugation (300 X g for 5 minutes), the pelleted sperm were
784
Coddington et al.
RESULTS Using the hemizona pellucida as a functional zona surface, the effect of PF was studied from patients with endometriosis. As shown in Figure lA, the number of sperm tightly bound was lower when the
Endometriotic PF decreased HZA binding
Fertility and Sterility
incubation was performed with PF from patients with endometriosis as compared with sperm incubated in media alone. Alterations in the number of sperm bound were more pronounced as the stage of endometriosis increased (mean ± SEM): stage I: 129 ± 30 (control) versus 96 ± 22 (test) (n = 6); stage II: 94 ± 67 (control) versus 25 ± 15 (test) (n = 3); and stage III: 171 ± 8 (control) versus 31 ± 15 (test) (n = 3). This fact was also confirmed when PF was used in the HZA control (Table 1). Variation in the number of sperm bound to the control zona is not unusual because the binding capacity of each zona may differ (7). Comparing the binding of each hemizona to itself, a hemizona index was calculated. For the case of an hemizona index equal to 100, equivalent binding for each would be noted. As the stage of endometriosis increased, the hemizona index decreased as pictured in Figure 1B (mean ± SEM): stage I: 72 ± 10 (n = 6); stage II: 58 ± 12 (n = 3); and stage III: 17 ± 8 (n = 3). Confirmation of the initial effect on the hemizona index was made using normal PF in the HZA control incubation. The results demonstrate a decreased binding of the sperm to the hemizona as the stage of endometriosis increased (mean ± SEM) (stage I: 47 ± 8, stage II: 22 ± 7; and stage III: 1 ± 0.6 [P < 0.03 between I versus II and III». DISCUSSION
Many aspects associated with endometriosis have been studied, but only a few have focused on sperm:
Table 1 Results of HZA in Which PF From Patients With Endometriosis and Normal Control Were Used in the Bioassay * No. of sperm bound test Stage I (n = 7) Stage II (n = 6) Stage III (n = 3)
No. of sperm bound control
Hemizona index
42
± 14
66 ± 13
47±8H
16
± 4
76 ± 16
22 ± 7t
0.3 ± 0.3
79 ±40
1 ± 0.6:j:
* Values are means ± SEM. t P < 0.03 for stage I versus stage II. :j: P < 0.03 for stage I versus stage III.
oocyte interaction. Interestingly, previous studies using the hamster sperm penetration assay (SPA) did not agree on the effect of PF of patients with endometriosis on the result of the SPA (3, 4). This is the first study that has sought to ascertain the effect of PF on the initial step of fertilization, tight binding of spermatozoa to the zona pellucida. Although the number of patients evaluated in the study is small, there appears to be an effect of the PF on zona:sperm interaction. This might represent a mechanism of infertility in some patients with endometriosis. It is not known whether changes are occurring at the level of the zona pellucida or at the level of sperm resulting in decreased binding, but the effect seems to be greater as the stage of endometriosis increases. Not only can the HZA evaluate the effects of endometriosis on sperm:oocyte interaction, but it might be useful as a marker to monitor this effect in treated patients as well.
~ BndomctriOlis
(A)
DConlroi
Acknowledgments. We gratefully acknowledge the expert assistance of Carlos Rotman, M.D., and Janice Hammond, B.S., for her technical assistance. We also thank Martha Wilson, B.A., for preparation of graphic materials, and Ms. Dara Willett-Leary for her editorial contribution. 81.,.1
(8)
REFERENCES
j
j Figure 1 (A), Average number of spermatozoa bound to the hemizona surface when exposed to PF from endometriosis patients. (B), Hemizona index (number of sperm bound to the hemizona from test/number of sperm bound to the hemizona from control X 100) for each stage of endometriosis.
Vol. 57, No.4, April 1992
1. Haney AF, Muscato JJ, Weinberg JB. Peritoneal fluid cell populations in infertility patients. Fertil Steril 1981;35:696-8. 2. Cyrop CH, Halme J. Peritoneal fluid environment and infertility. In: Wallach EE, Kempers RD, editors. Modern trends in infertility and contraception control. Boca Raton (FL): Year Book Medical Publishers, Inc., 1988:343-51. 3. Chacho KJ, Chacho MS, Anderson PJ, Scommegna A. Peritoneal fluid in patients with and without endometriosis. Prostanoids and macrophages and their effect on the spermatozoa penetration assay. Am J Obstet Gynecol 1986;154: 1290-9.
Coddington et al.
Endometriotic PF decreased HZA binding
785
4. Halme J, Hall JL. Effect of pelvic fluid from endometriosis patients on human sperm penetration of zona-free hamster ova. Fertil Steril 1982;37:573-6. 5. Sueldo CE, Lambert H, Steinleitner A, Rathwick G, Swanson J. The effect of peritoneal fluid from patients with endometriosis on murine sperm-oocyte interaction. Fertil Steril 1987;48:697-9. 6. Morcos RN, Gibbons WE, Findley WE. Effect of peritoneal fluid on in vitro cleavage of 2-cell mouse embryos: possible role in infertility associated with endometriosis. Fertil Steril 1985;44:678-83. 7. Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen GO. The hemizona assay (HZA): devel-
786
Coddington et aI.
opment of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril1988;49:688-97. 8. Oehninger S, Coddington CC, Scott R, Franken DR, Burkman LJ, Acosta AA, et al. Hemizona assay (HZA): assessment of sperm dysfunction and prediction of in vitro fertilization outcome. Fertil Steril1989;51:665-70. 9. Coddington CC, Fulgham DL, Alexander NJ, Johnson 0, Herr JC, Hodgen GO. Sperm bound to the zona pellucida in the hemizona assay demonstrate acrosome reaction when stained with T-6 antibody. Fertil Steril1990;54:504-8. 10. The American Fertility Society classification of endometriosis. Fertil Steril1985;43:351-2.
Endometriotic PF decreased HZA binding
Fertility and Sterility
Vol. 57, No.4, April 1992
Copyright" 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Peritoneal fluid from patients with endometriosis decreases sperm binding to the zona pellucida in the hemizona assay: a preliminary report*
Charles C. Coddington, M.D.t:!:§ Sergio Oehninger, M.D.t Dean S. Cunningham, Ph.D., L.C.D.R., M.C., U.S.N.:!:
Keith Hansen, L.C.D.R., M.C., U.S.N.R.:!: Carlos E. Sueldo, M.D. II Gary D. Hodgen, Ph.D.t
The Jones Institute for Reproductive Medicine, Eastern Virginia Medical Schoo~ Norfolk; Portsmouth Naval Hospita~ Portsmouth, Virginia; and Valley Medical Center, University of California, Fresno, California
Objective: To evaluate the effect of peritoneal fluid (PF) from patients with endometriosis on gamete interaction under hemizona assay (HZA) conditions. Design: The HZA was used to study the effect of PF from patients with endometriosis on sperm binding to the zona pellucida using media and normal PF as controls. Setting: The patients were collected from a university hospital infertility clinic. Patients: Peritoneal fluid from 16 women being evaluated for infertility or sterilization who were found to have endometriosis at surgery was used. Three normal patients, who were being sterilized, had PF that was used as a control. Results: Results suggest that there is a significant reduction in the number of tightly bound sperm to the zona surface in endometriosis specimens as reflected in the hemizona index and that this effect is directly related to the stage of the disease. Conclusions: Although the sample is small, this methodology may help to elucidate one of the mechanisms responsible for endometriosis-associated infertility. Fertil SteriI1992;57:783-6 Key Words: Endometriosis, peritoneal fluid, hemizona assay, sperm:oocyte binding, sperm function
Endometriosis is a disease of the female pelvis that is associated with pelvic pain, irregular vaginal bleeding, and infertility. Although mechanical factors can be related to an etiology of infertility with Received July 11, 1991; revised and accepted December 3, 1991.
* Presented in part at the 46th Annual Meeting of The American Fertility Society, Washington, D.C., October 13 to 18, 1990. t The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School. Portsmouth Naval Hospital. § Reprint requests: Charles C. Coddington, M.D., The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia 23507. II Valley Medical Center, University of California.
*
Vol. 57, No.4, April 1992
more severe disease, little is known about the pathophysiology of infertility in the less advanced stages of the disease. Several authors have demonstrated that there are increased numbers of macrophages in the peritoneal cavity of patients with endometriosis (1, 2), but others have questioned this finding (3). Many studies have been performed to evaluate the pelvic cavity environment, but there does not appear to be a clear marker to relate to infertility (1-6). Little information has accumulated about gamete interaction. Studies have been performed to evaluate the effect of peritoneal fluid (PF) on the ability of sperm to penetrate hamster zonafree oocytes (3,4), but the results are conflicting. It has been reported that PF from patients with en-
Coddington et aI.
Endometriotic PF decreased HZA binding
783
dometriosis caused decreased fertility in the mouse model (5) and also caused toxic effects on mouse embryo cleavage (6). Currently, there is not a method that evaluates the effect of PF on the steps of fertilization that occur naturally. The hemizona assay (HZA) has been developed to evaluate tight binding of the sperm to the zona pellucida, a critical event leading to fertilization (7). Studies have confirmed that fertile patients have increased numbers of sperm bound to the zona (7), and increased success in in vitro fertilization has been associated with higher numbers of sperm bound (8). Additionally, sperm bound to the hemizona have undergone the expected acrosome reaction as dem0nstrated by the T-6/antibody with immunofluorescent staining (9). Thus, the HZA is a unique, homologous bioassay by which gamete interaction can be evaluated. Our purpose was to determine the effect of PF from patients with endometriosis on sperm-oocyte interaction under HZA conditions.
washed a second time. The second pellet was overlayed with 0.5 mL of F-10 medium and incubated (37°C, 5% CO2 in air) to achieve a swim-up separation. After 1 hour of incubation, the sperm supernatant was removed and used for testing of hemizona binding. Peritoneal Fluid Peritoneal fluid was obtained from the abdominal cavity of 16 regular cycling women with endometriosis and 3 normal women (controls) who were on days 9 through 15 of their menstrual cycle. Their ages ranged from 25 to 30, and the indication for surgery was for tubal ligation or evaluation of infertility. Endometriosis staging was performed according to current American Fertility Society criteria (10). The fluid was obtained and centrifuged at 200 X g for 10 minutes to remove cellular contents. Supernatant was pipetted into plastic storage tubes and frozen at -4°C. At time of use, the fluid was thawed and warmed before being used in the HZA in a 5:1 dilution with medium containing sperm.
MATERIALS AND METHODS Protocol for HZA Semen Analysis Semen from three fertile donors who had been previously extensively tested in an HZA was used in the studies. The basic semen evaluation was performed using an automated, computerized system (Cell Soft Semen Analyzer, Cryo Resources Ltd., New York, NY). Details of these procedures have been described previously (7). Oocyte Handling and Zona Cutting Human oocytes were obtained from postmortem ovarian tissue. The ovarian tissue was placed on ice after excision, dissected, and minced in phosphatebuffered saline (7). Narishige micromanipulators (Narishige, Tokyo, Japan) mounted on a phase-contrast inverted microscope (Nikon Diaphot, Garden City, NY) were used for equally bisecting the oocytes. The cutting procedures have previously been published (7, 8).
A 100-J'L droplet ofthe sperm suspension (500,000 motile sperm/mL after swim-up) was placed in a Petri dish under oil. In initial experiments, one hemizona was placed in a droplet with sperm that had been exposed to the PF being studied (1:5) (test), whereas the matching hemizona was incubated with a droplet ofsperm exposed to Ham's F-10 and 7.5% fetal cord serum (control). For the second set of experiments, PF from patients with a normal pelvis was used in the control media in the same dilution. The gametes were coincubated for 4 hours (37°C, 5 % CO2 in air). Each hemizona was then removed and rinsed using a fine glass pipette in medium to separate loosely associated sperm (7). The number oftightly bound sperm was counted. Average values were obtained.for number of sperm bound and hemizona index. The hemizona index is the number of sperm bound from test/number of sperm bound from control X 100 (7). A paired t-test was used to compare the hemizona index data.
Sperm Handling for the HZA An aliquot of semen (0.5 mL) was diluted with 1 mL of Ham's F-10 culture medium (GIBCO, Grand Island NY), supplemented with 7.5% heat-inactivated human fetal cord serum. After centrifugation (300 X g for 5 minutes), the pelleted sperm were
784
Coddington et al.
RESULTS Using the hemizona pellucida as a functional zona surface, the effect of PF was studied from patients with endometriosis. As shown in Figure lA, the number of sperm tightly bound was lower when the
Endometriotic PF decreased HZA binding
Fertility and Sterility
incubation was performed with PF from patients with endometriosis as compared with sperm incubated in media alone. Alterations in the number of sperm bound were more pronounced as the stage of endometriosis increased (mean ± SEM): stage I: 129 ± 30 (control) versus 96 ± 22 (test) (n = 6); stage II: 94 ± 67 (control) versus 25 ± 15 (test) (n = 3); and stage III: 171 ± 8 (control) versus 31 ± 15 (test) (n = 3). This fact was also confirmed when PF was used in the HZA control (Table 1). Variation in the number of sperm bound to the control zona is not unusual because the binding capacity of each zona may differ (7). Comparing the binding of each hemizona to itself, a hemizona index was calculated. For the case of an hemizona index equal to 100, equivalent binding for each would be noted. As the stage of endometriosis increased, the hemizona index decreased as pictured in Figure 1B (mean ± SEM): stage I: 72 ± 10 (n = 6); stage II: 58 ± 12 (n = 3); and stage III: 17 ± 8 (n = 3). Confirmation of the initial effect on the hemizona index was made using normal PF in the HZA control incubation. The results demonstrate a decreased binding of the sperm to the hemizona as the stage of endometriosis increased (mean ± SEM) (stage I: 47 ± 8, stage II: 22 ± 7; and stage III: 1 ± 0.6 [P < 0.03 between I versus II and III». DISCUSSION
Many aspects associated with endometriosis have been studied, but only a few have focused on sperm:
Table 1 Results of HZA in Which PF From Patients With Endometriosis and Normal Control Were Used in the Bioassay * No. of sperm bound test Stage I (n = 7) Stage II (n = 6) Stage III (n = 3)
No. of sperm bound control
Hemizona index
42
± 14
66 ± 13
47±8H
16
± 4
76 ± 16
22 ± 7t
0.3 ± 0.3
79 ±40
1 ± 0.6:j:
* Values are means ± SEM. t P < 0.03 for stage I versus stage II. :j: P < 0.03 for stage I versus stage III.
oocyte interaction. Interestingly, previous studies using the hamster sperm penetration assay (SPA) did not agree on the effect of PF of patients with endometriosis on the result of the SPA (3, 4). This is the first study that has sought to ascertain the effect of PF on the initial step of fertilization, tight binding of spermatozoa to the zona pellucida. Although the number of patients evaluated in the study is small, there appears to be an effect of the PF on zona:sperm interaction. This might represent a mechanism of infertility in some patients with endometriosis. It is not known whether changes are occurring at the level of the zona pellucida or at the level of sperm resulting in decreased binding, but the effect seems to be greater as the stage of endometriosis increases. Not only can the HZA evaluate the effects of endometriosis on sperm:oocyte interaction, but it might be useful as a marker to monitor this effect in treated patients as well.
~ BndomctriOlis
(A)
DConlroi
Acknowledgments. We gratefully acknowledge the expert assistance of Carlos Rotman, M.D., and Janice Hammond, B.S., for her technical assistance. We also thank Martha Wilson, B.A., for preparation of graphic materials, and Ms. Dara Willett-Leary for her editorial contribution. 81.,.1
(8)
REFERENCES
j
j Figure 1 (A), Average number of spermatozoa bound to the hemizona surface when exposed to PF from endometriosis patients. (B), Hemizona index (number of sperm bound to the hemizona from test/number of sperm bound to the hemizona from control X 100) for each stage of endometriosis.
Vol. 57, No.4, April 1992
1. Haney AF, Muscato JJ, Weinberg JB. Peritoneal fluid cell populations in infertility patients. Fertil Steril 1981;35:696-8. 2. Cyrop CH, Halme J. Peritoneal fluid environment and infertility. In: Wallach EE, Kempers RD, editors. Modern trends in infertility and contraception control. Boca Raton (FL): Year Book Medical Publishers, Inc., 1988:343-51. 3. Chacho KJ, Chacho MS, Anderson PJ, Scommegna A. Peritoneal fluid in patients with and without endometriosis. Prostanoids and macrophages and their effect on the spermatozoa penetration assay. Am J Obstet Gynecol 1986;154: 1290-9.
Coddington et al.
Endometriotic PF decreased HZA binding
785
4. Halme J, Hall JL. Effect of pelvic fluid from endometriosis patients on human sperm penetration of zona-free hamster ova. Fertil Steril 1982;37:573-6. 5. Sueldo CE, Lambert H, Steinleitner A, Rathwick G, Swanson J. The effect of peritoneal fluid from patients with endometriosis on murine sperm-oocyte interaction. Fertil Steril 1987;48:697-9. 6. Morcos RN, Gibbons WE, Findley WE. Effect of peritoneal fluid on in vitro cleavage of 2-cell mouse embryos: possible role in infertility associated with endometriosis. Fertil Steril 1985;44:678-83. 7. Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen GO. The hemizona assay (HZA): devel-
786
Coddington et aI.
opment of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril1988;49:688-97. 8. Oehninger S, Coddington CC, Scott R, Franken DR, Burkman LJ, Acosta AA, et al. Hemizona assay (HZA): assessment of sperm dysfunction and prediction of in vitro fertilization outcome. Fertil Steril1989;51:665-70. 9. Coddington CC, Fulgham DL, Alexander NJ, Johnson 0, Herr JC, Hodgen GO. Sperm bound to the zona pellucida in the hemizona assay demonstrate acrosome reaction when stained with T-6 antibody. Fertil Steril1990;54:504-8. 10. The American Fertility Society classification of endometriosis. Fertil Steril1985;43:351-2.
Endometriotic PF decreased HZA binding
Fertility and Sterility