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Obstetrics & Gynecology 2001;98:668-673
© 2001 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Macrophage Secretory Products and Sperm Zona Pellucida Binding

Barbara M. Faber, MD, Nasser Chegini, PhD, Mary C. Mahony, PhD and Charles C. Coddington, III, MD

From the Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia; and the Department of Obstetrics and Gynecology, University of Florida, Gainesville, Florida.

Address reprint requests to: Barbara M. Faber, MD, 1300 North 12th Street, Suite 520, Phoenix, AZ 85006; E-mail: bfaber{at}arizonarms.com.


   ABSTRACT
TOP
ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE: To determine if exposure of human gametes to macrophage secretory products reduces sperm binding to the zona pellucida, and to determine which cytokine(s) may be responsible for this effect.

METHODS: A human macrophage cell line was cultured and either activated with lipopolysaccharide for 2 hours and then washed or left unactivated. Culture-conditioned media from activated or unactivated cells was used in hemizona assay. Hemizonae were incubated with sperm suspended in culture medium from either unactivated macrophages or activated macrophages, with the matching hemizona incubated with sperm suspended in control medium. Matching hemizonae were incubated with sperm suspended in unactivated macrophage medium paired with sperm suspended in activated macrophage culture medium. Conditioned medium from activated macrophages was found to have elevated levels of tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin-1ß, and transforming growth factor-ß, therefore, gametes were also exposed to these cytokines followed by the hemizona assay. After each incubation, the number of sperm tightly bound to the outer surface of each hemizona was determined.

RESULTS: Exposure of gametes to activated and unactivated macrophage culture-conditioned media significantly decreases sperm binding to the zona pellucida, with medium from activated macrophages inducing the greatest effect (P < .05). Exposure of sperm to TNF-{alpha} significantly impaired sperm binding (P < .05), whereas other cytokines tested had no effect.

CONCLUSION: These results suggest that macrophage secretory products in the basal and activated state may be a factor in endometriosis-associated infertility through the interference of sperm binding to the zona pellucida, and that TNF-{alpha} is a key cytokine responsible for this effect.

Endometriosis is characterized by the presence and growth of ectopic endometrial tissue outside the uterine cavity. Retrograde menstruation is thought to be the mechanism by which endometrial fragments are transported into the pelvis where their ultimate proliferation at ectopic sites causes pelvic pain, dyspareunia, and infertility.1–3 An association of endometriosis with infertility is supported by the finding that women with infertility were more likely to have endometriosis diagnosed at laparoscopy when compared with fertile controls.4 The mechanisms involved in the pathophysiology of endometriosis and the cause-and-effect relationship between endometriosis and infertility remain unclear and, at best, controversial in less-advanced disease.

The presence of an unfavorable environment, created by an immune- or inflammation-related condition in the peritoneal cavity as well as in the reproductive tract (ie, ovary, fallopian tube, and endometrium), has been proposed to participate in pathophysiology of endometriosis, including endometriosis-associated infertility.5–28 This environment consists of the peritoneal fluid, its cellular constituents, and their secretory products. Peritoneal fluid from patients with endometriosis has been shown to have a deleterious effect on oocyte pickup, sperm motility, sperm–oocyte interaction, and embryonic development.8–10,21 Peritoneal fluid of infertile women with endometriosis has also been shown to have an increased number of activated macrophages and elevated levels of macrophage-derived growth factors and cytokines, such as interleukins (IL) IL-1ß, IL-6, IL-8, tumor necrosis factor-{alpha} (TNF-{alpha}), and transforming growth factor-ß (TGF-ß).6,7,10–12,15,16,19,23,24,27 As a potential mechanism of endometriosis-associated infertility, secretory products of the activated macrophage have been shown to inhibit in vitro fertilization and early embryonic development in mice, possibly due to elevated production and cytotoxic effects of cytokines such as TNF-{alpha} and ILs.22

We previously reported that peritoneal fluid of women with endometriosis impairs sperm binding to the zona pellucida in the hemizona assay, a bioassay by which gamete interaction can be evaluated.20 In this study, the effect on sperm binding was greater as the stage of the endometriosis increased. However, which gamete was affected, the identity of the molecule(s), and the cellular origin of the factors in peritoneal fluid affecting sperm binding were not defined. To investigate this further, we hypothesize that activated macrophage–derived factors interfere with sperm zona pellucida binding, the earliest step in gamete interaction, and therefore, cause endometriosis associated infertility. To test this hypothesis, culture-conditioned media were prepared from lipopolysaccharide-activated and unactivated macrophages and then used in the hemizona assay. Hemizonae and sperm were exposed to conditioned medium from activated cells, unactivated cells, or control medium to test sperm binding to the zona pellucida. In addition, because activated macrophages express elevated levels of several cytokines, including TNF-{alpha}, IL-1ß, and TGF-ß, we investigated whether sperm zona pellucida binding is directly affected by exposure to these cytokines, thus in part determining which factors may be responsible for this effect.


   MATERIALS AND METHODS
TOP
ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All the materials and supplies for these experiments were purchased from commercial sources as previously described.20,22,29 An established human macrophage cell line, U937, was purchased from American Type Culture Collection (Rockville, MD). Lipopolysaccharide, Limulus amebocyte lysate kit, all the culture media, fetal bovine serum, and human serum were purchased from Sigma Chemical (St. Louis, MO). Human specific TNF-{alpha}, IL-1ß, and TGF-ß1 enzyme-linked immunosorbent assay (ELISA) kits and human recombinant TNF-{alpha}, IL-1ß, and TGF-ß were purchased from R & D Systems (Minneapolis, MN).

The cells were grown in 75-cm2 flasks in RPMI 1640 supplemented with 10% fetal bovine serum for 4–7 days. The cells were collected, washed, and resuspended at 106 cells/mL in serum-free medium. They were then either activated with 12.5 µg/mL of lipopolysaccharide for 1 hour, or kept unactivated in serum-free media as described previously.22 After the incubation, lipopolysaccharide-activated macrophages were washed several times with control medium until endotoxin was undetectable in the medium. The lipopolysaccharide-activated and unactivated cells were then cultured with RPMI 1640 supplemented with 0.5% fetal bovine serum for 24 hours. Activation of the macrophages resulted in the characteristic adherence to the surface of the culture flask. After the incubation, the media from activated and unactivated macrophages was collected and stored at -80 C until it was used in hemizona assay.

Oocytes and sperm used in this study were obtained from the IVF Laboratory at the Jones Institute for Reproductive Medicine (Norfolk, VA) after obtaining approval of the study protocol by the Institutional Review Board. Patients from whom the oocytes were obtained were fully informed and gave written consent. Oocytes were immature, noninseminated prophase 1, or mature, inseminated but unfertilized. Oocytes were kept in balanced salt solution at 4 C and bisected into matching hemizonae using Narishige micromanipulators (Narishige, Tokyo, Japan) mounted on a phase-contrast inverted microscope (Diaphot; Nikon, Garden City, NY) before their use in the hemizona assay according to procedures previously described.20,29 Semen was provided by donors whose sperm exhibited consistently good binding capacity in the hemizona assay.

Liquefied semen was diluted with Ham’s F-10 (Gibco Laboratories, Grand Island, NY) supplemented with 5% human serum albumin and centrifuged to remove the seminal plasma. The motile sperm fraction was collected by a 1-hour swim-up procedure. The supernatant was removed, and the motile sperm concentration was adjusted to 15 x 106/mL. Sperm were then collected by centrifugation and suspended in activated or unactivated macrophage culture–conditioned media or control medium (RPMI 1640 supplemented with 0.5% fetal bovine serum) at a motile sperm concentration of 2 x 106/mL.

The hemizona assay was then performed as described previously,20,29 using 100-µL droplets of sperm suspended in activated macrophage–culture conditioned medium, unactivated macrophage culture–conditioned medium, or control medium. One hemizona was placed in medium with the matching hemizona placed in different media as follows: control medium versus unactivated macrophage–conditioned medium; control medium versus activated macrophage–conditioned medium, and unactivated macrophage–conditioned medium versus activated macrophage–conditioned medium.

The gametes were incubated under oil for 4 hours at 37 C and 5% CO2 in a humidified atmosphere. After the incubation, the hemizonae were rinsed five times to remove loosely attached sperm, and the number of sperm tightly bound to the outer zona surface were counted under phase contrast microscopy (x200). The hemizona assay was also preformed using decreasing concentrations of activated or unactivated macrophage culture–conditioned media diluted with control medium and compared with control medium.

The level of TNF-{alpha}, IL-1ß, and TGF-ß1 in macrophage–conditioned media was determined using ELISA kits with limited detection of 4.4, 1.0, and 7 pg/mL, respectively, according to recommended procedures.

In the next experiment, a 1-hour swim-up procedure was again used, and the motile fraction of sperm was exposed to human recombinant TNF-{alpha}, IL-1ß, and TGF-ß at concentrations equal to levels found in activated macrophage culture–conditioned media. Hemizonae were then exposed to sperm suspended in each cytokine, while the matching hemizonae were exposed to Ham’s F-10 medium supplemented with 0.5% human serum albumin. Once it was determined that TNF-{alpha} impaired sperm binding, varying concentrations of this cytokine were used to establish its dose-dependent action in the hemizona assay.

Comparisons between the effect of activated and unactivated macrophage culture–conditioned media, and the effect of TNF-{alpha} on tight sperm–zona binding were made using the number of tightly bound sperm or the hemizona index. The hemizona index is calculated as follows:


Inherent oocyte variability accounts for differences in sperm binding for a particular medium between hemizona assay pairings. The data were expressed as the mean ± standard error of the mean and were statistically analyzed using a t test and analysis of variance with post hoc comparison testing with P < .05 considered to be significant.


   RESULTS
TOP
ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Exposure of the gametes during the hemizona assay to either activated or unactivated macrophage–conditioned media resulted in a significant decrease in sperm binding to the zona pellucida compared with control medium (Figure 1Go). Such an inhibitory effect on sperm zona pellucida binding was observed to be more significant using activated macrophage–conditioned medium compared with unactivated macrophage–conditioned medium (Figure 1Go). Because the macrophages’ secretory products released into the culture-conditioned media are responsible for the altered sperm binding to zona pellucida, we expect to see a reduction in their inhibitory action after serial dilution. Using increasing dilutions of activated and unactivated macrophage–conditioned media, we found an increase in tight binding of sperm to the zona pellucida, expressed as the hemizona index (Figures 2Go and 3Go).



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  		Figure 1. The effect of culture-conditioned media from lipopolysaccharide-activated (AM-CM) and unactivated (M-CM) macrophage on sperm–zona pellucida binding compared with control culture medium (RPMI with 0.5% fetal bovine serum) (C-CM). The number of hemizona pairings used in C-CM vs M-CM was 18, C-CM vs AM-CM was 18, and M-CM vs AM-CM was 11. The result is expressed as the number of sperm bound to the hemizona surface and was statistically analyzed with a paired t test. In a vs b, P = .001; in c vs d, P < .001; and in e vs f, P =.001.

Faber. Fertilization and Endometriosis. Obstet Gynecol 2001.

 


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  		Figure 2. The effect of decreasing concentrations of activated macrophage–conditioned medium on sperm–zona pellucida binding expressed as a hemizona index. Two hemizona pairings were used for 1:1000 dilution, whereas three hemizona pairings were used for the other dilutions, and 18 hemizona pairings were used for undiluted conditioned media. The results were statistically analyzed with analysis of variance and post hoc multiple comparison testing. * different from ** P < .05.

Faber. Fertilization and Endometriosis. Obstet Gynecol 2001.

 


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  		Figure 3. The effect of decreasing concentrations of unactivated macrophage–conditioned medium on sperm–zona pellucida binding expressed as the hemizona index. Three hemizona pairings were used for assay in 1:100 dilution, five for 1:20, and 18 for undiluted conditioned media. The results were statistically analyzed with analysis of variance and post hoc multiple comparison testing. * different from **P < .05.

Faber. Fertilization and Endometriosis. Obstet Gynecol 2001.

 
The activated macrophage culture–conditioned medium had a significantly higher level of IL-1ß, TNF-{alpha}, and TGF-ß compared with the unactivated macrophage culture–conditioned medium (Figure 4Go). However, most of the TGF-ß detected in activated and unactivated macrophage–conditioned medium was in latent form. Control medium (RPMI containing 0.5% fetal bovine serum) unexposed to macrophages did not have any detectable levels of IL-1ß and TNF-{alpha}, but it contained a low level of TGF-ß that ranged from 80 to 100 g/mL. We found that IL-1ß or TGF-ß at concentrations detected in the media had no significant effect on binding of sperm to the zona pellucida (data not shown). In contrast, tight sperm binding to the zona pellucida, the hemizona index, was significantly decreased after exposure to TNF-{alpha} (Figure 5Go). The effect of TNF-{alpha} on hemizona index was dose dependent and occurred at very low concentrations of this cytokine (Figure 5Go).



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  		Figure 4. The level of interleukin-1ß (IL-1ß), tumor necrosis factor-{alpha} (TNF-{alpha}), and transforming growth factor-ß (TGF-ß1) in culture-conditioned media from lipopolysaccharide-activated and unactivated macrophages. Results were statistically analyzed with unpaired t test. In a vs b, P < .05; c vs d, P < .05; e vs f, P < .05.

Faber. Fertilization and Endometriosis. Obstet Gynecol 2001.

 


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  		Figure 5. The effect of tumor necrosis factor-{alpha} (TNF-{alpha}) at different concentrations on sperm–zona pellucida binding expressed as the hemizona index. The number of hemizona pairings used in these assays were n = 2, 4, 6, and 3 for each dilution, respectively. The results were statistically analyzed with analysis of variance and post hoc multiple comparison testing. * different from ** P < .05.

Faber. Fertilization and Endometriosis. Obstet Gynecol 2001.

 

   DISCUSSION
TOP
ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An alteration of the immune/inflammatory response in the reproductive tract and peritoneal environment is considered to play a major role in the pathophysiology of endometriosis and thus endometriosis-associated infertility. Under normal conditions, immune and inflammatory cells are present in the peritoneal fluid as well as in ovarian, oviductal, and endometrial tissues. These cells—especially macrophages in an activated state—express, synthesize, and release many substances, including those with growth-promoting and cytotoxic properties.16 Many of these secretory products have beneficial and regulatory effects on a variety of biologic activities, as well as on oocytes, spermatozoa, and embryos, during transportation, fertilization, and the preimplantation period.18,19 However, the presence of an unfavorable environment, such as an immune/inflammatory reaction in these tissues or in the peritoneal cavity, as observed in women with endometriosis, may ultimately lead to failure in oocyte maturation, ovulation, fertilization, and early embryonic development.13,16–18,21,22 Whether such inflammatory reactions occur at one site, or at multiple sites simultaneously, and whether they occur before or immediately after the development of endometriosis, is not known.

In women with endometriosis, the peritoneal fluid volume and the number of peritoneal macrophages have been reported to be significantly increased when compared with women without disease, possibly as a result of an intraperitoneal inflammatory response to the presence of ectopic endometrial implants.6,15,16 In the oviduct, to which peritoneal macrophages have access through peritoneal fluid, macrophages are also present in the tubal subepithelial region.18,19 It is therefore essential to consider macrophages and the effects of their secretory products in pathogenesis of endometriosis-associated infertility.

The tight binding of the spermatozoa to the zona pellucida is a critical initial event in the fertilization process. To further explore the potential implication of activated macrophages and their secretory products on this event, we used a well-established and recognized assay to assess sperm–zona pellucida binding. We found that the sperm–zona pellucida binding is severely inhibited after exposure to both conditioned medium prepared from activated and unactivated macrophages, with greater potency obtained with media from activated macrophages. It has been established that the number of macrophages and the level of their secretory products released into the peritoneal fluid is increased in women with endometriosis, and that these factors may play a key role in endometriosis-associated infertility. Our results provide evidence that the detrimental action of macrophage secretory products, particularly in the activated state, occurs at the earliest and most critical stage through a mechanism that involves the interference of sperm–zona pellucida binding. We have shown previously that peritoneal fluid from patients with endometriosis impairs sperm binding to the zona pellucida.20 The results of our present study provide evidence that the macrophage secretory products are responsible for the impaired sperm binding to the zona pellucida, induced by peritoneal fluid from patient’s with endometriosis.

The activated macrophages express elevated levels of various cytokines, including TGF-ß, IL-1{alpha}, IL-1ß, and TNF-{alpha}. We have reported previously that co-incubation of the activated macrophages or exposure to their secretory products, in a stage-specific manner, directly alters early embryonic development and trophoblastic outgrowth.22 Our present data indicate that TNF-{alpha} released by macrophages is, at least in part, responsible for impaired sperm–zona pellucida binding. The effect of TNF-{alpha} occurred in a concentration-dependent manner and was found to be effective at inhibiting tight sperm binding even at low concentrations. It has been reported that both basal and lipopolysaccharide-stimulated peritoneal macrophage of women with endometriosis have elevated production of TNF-{alpha}, as well as other cytokines, when compared with macrophages from fertile controls.24 Interestingly, the level of TNF-{alpha} produced by lipopolysaccharide-stimulated peritoneal macrophages reported in this study for infertile endometriosis patients is comparable to the concentration we found to impair sperm–zona pellucida binding. Furthermore, we found that the concentration of TNF-{alpha} at which the impairment of sperm binding was dissipated is similar to the stimulated level of TNF-{alpha} produced by macrophages of the fertile patients in that study.

The inhibitory effect of TNF-{alpha} on sperm binding was not seen at the concentration of TNF-{alpha} detected in unactivated macrophage–conditioned medium, suggesting that other factors must also be responsible for this effect.

Previous reports have implicated a role for IL-1ß and TGF-ß1 in endometriosis-associated abnormalities,22,26,27 however, we did not find any significant impairment of sperm–zona pellucida binding induced by IL-1ß or TGF-ß.

In summary, the results of this study provide further evidence that macrophage secretory products, particularly TNF-{alpha}, may participate in endometriosis-associated infertility through the interference of sperm–zona pellucida binding. Further studies are required to determine the detailed molecular mechanisms involved in impairment of sperm–zona pellucida binding induced by TNF-{alpha} and possibly other macrophage secretory products; thus leading to new clinical interventions for better management of patients with endometriosis-associated infertility.


   Footnotes
 
Dr. Faber is the recipient of the American College of Obstetrics and Gynecology Mead Johnson Bristol-Myers Squibb Research Fellowship in Obstetrics and Gynecology.

PII S0029-7844(01)01510-1

Received January 16, 2001. Received in revised form June 6, 2001. Accepted June 15, 2001.


   REFERENCES
TOP
ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Metzger DA, Haney AF. Endometriosis: Etiology and pathophysiology of infertility. Clin Obstet Gynecol 1988; 31:801–12.[Medline]

2. Haney AF. Etiology and histogenesis of endometriosis. Prog Clin Biol Res 1990;323:1–14.

3. Halme J, Hammond MG, Hulka JF, Raj SG, Talbert LM. Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 1984;64: 151–4.[Abstract/Free Full Text]

4. Strathy JH, Monlgaard CA, Coulam CB, Melton LJ. Endometriosis and infertility: A laparoscopic study of endometriosis among fertile and infertile women. Fertil Steril 1982;38:667–72.[Medline]

5. Syrop CH, Halme J. Peritoneal fluid environment and infertility. In: Wallach EE, Kempers RD, eds. Modern trends in infertility and contraception control. Boca Raton, FL: Year Book Medical Publishers, Inc., 1988:343–51.

6. Koninckx PR, Kennedy SH, Barlow DH. Endometriotic disease: The role of peritoneal fluid. Hum Reprod Update 1998;4:741–51.[Abstract/Free Full Text]

7. Ramey JW, Archer DF. Peritoneal fluid: Its relevance to the development of endometriosis. Fertil Steril 1993;60: 1–14.[Medline]

8. Suginami H, Yanko K. An ovum capture inhibitor (OCI) in endometriosis peritoneal fluid: An OCI-membrane responsible for fimbrial failure of ovum capture. Fertil Steril 1988;50:648–53.[Medline]

9. Aeby TC, Huang T, Nakayama RT. The effect of peritoneal fluid from patients with endometriosis on human sperm function in vitro. Am J Obstet Gynecol 1996;174: 1779–85.[Medline]

10. Taketani Y, Tong-Ming K, Mizuno M. Comparison of cytokine levels and embryo toxicity in peritoneal fluid in infertile women with untreated or treated endometriosis. Am J Obstet Gynecol 1992;167:265–70.[Medline]

11. Koyama N, Matsuura K Okamura H. Cytokines in the peritoneal fluid of patient with endometriosis. Int J Gynecol Obstet 1993;43;45–50.[Medline]

12. Ryan IP, Tsen JF, Schriock ED, Khorram O, Landers DV, Taylor RN. Interleukin-8 concentrations are elevated in peritoneal fluid of women with endometriosis. Fertil Steril 1995;63:929–32.[Medline]

13. Pellicer A, Albert C, Garrido N, Navarro J, Remohi J, Simon C. The pathophysiology of endometriosis-associated infertility: Follicular environment and embryo quality. J Reprod Fertil Suppl 2000;55:109–19.[Medline]

14. Pellicer A, Albert C, Mercader A, Bonilla-Musoles F, Remohi J, Simon C. The follicular and endocrine environment in women with endometriosis: Local and systemic cytokine production. Fertil Steril 1998;70:425–31.[Medline]

15. Rier SE, Yeaman GR. Immune aspects of endometriosis: Relevance of the uterine mucosal immune system. Semin Reprod Endocrinol 1997;15:209–20.[Medline]

16. Oral E, Arici A. Peritoneal growth factors and endometriosis. Semin Reprod Endocrinol 1996;14:257–67.[Medline]

17. Carlberg M, Nejaty J, Froysa B, Guan Y, Soder O, Bergqvist A. Elevated expression of tumor necrosis factor alpha in cultured granulosa cells from women with endometriosis. Hum Reprod 2000;15:1250–5.[Abstract/Free Full Text]

18. Chegini N. Oviductal-derived growth factors and cytokines: Implication in preimplantation. Semin Reprod Endocrinol 1996;14:219–29.[Medline]

19. Chegini N, Williams RS. Macrophages and macrophage-derived factors in normal reproductive tissues and endometriosis. In: Diamond M, Osteen K, eds. Endometrium and endometriosis. Cambridge: Malden Blackwell Sciences Inc., 1997;140–5.

20. Coddington CC, Oehninger S, Cunningham DS, Hansen K, Sueldo CR, Hodgen GD. Peritoneal fluid from patients with endometriosis decreases sperm binding to the zona pellucida in the hemizona assay (HZA): A preliminary report. Fertil Steril 1992;57:783–6.[Medline]

21. Illera MJ, Juan L, Stewart CL, Cullinan E, Ruman J, Lessey BA. Effect of peritoneal fluid from women with endometriosis on implantation in the mouse model. Fertil Steril 2000;74:41–8.[Medline]

22. Juneja SC, Pfeifer TL, Tang X-M, Williams RS, Chegini N. Modulation of mouse sperm-egg interaction, early embryonic development and trophoblastic outgrowth by activated and unactivated macrophages. Endocrine 1995; 3:69–79.

23. Harada T, Enatsu A, Mitsunari M, Nagano Y, Ito M, Tsudo T, et al. Role of cytokines in progression of endometriosis. Gynecol Obstet Invest 1999;47(Suppl. 1):34–9; discussion 39–40.

24. Rana N, Braun DP, House R, Gebel H, Rotman C, Dmowski WP. Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. Fertil Steril 1996;65:925–30.[Medline]

25. Iwabe T, Harada T, Tsudo T, Nagano Y, Yoshida S, Tanikawa M, et al. Tumor necrosis factor-alpha promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression. J Clin Endocrinol Metab 2000;85:824–9.[Abstract/Free Full Text]

26. Chegini N, Gold LI, Williams RS. Localization of transforming growth factor beta isoforms TGF-ß1, TGF-ß2, and TGF-ß3 in surgically induced endometriosis in the rat. Obstet Gynecol 1994;83:455–61.[Abstract/Free Full Text]

27. Oosterlynck DJ, Meuleman C, Waer M, Koninckx PR. Transforming growth factor-beta activity is increased in peritoneal fluid from women with endometriosis. Obstet Gynecol 1994;83:287–92.[Abstract/Free Full Text]

28. Juneja SC, Chegini N, Williams RS, Ksander GA. Ovarian intrabursal administration of transforming growth factor-ß1 inhibits follicle rupture in gonadotropin-primed mouse. Biol Reprod 1996;55:1444–51.[Abstract]

29. Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen GD. The hemizona assay (HZA); development of a diagnostic test for the binding of human spermatozoa to the hum hemizona pellucida to predict fertilization potential. Fertil Steril 1988;49:688–97.[Medline]





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