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Follicular fluid prostaglandins in endometriosis and ovarian hyperstimulation*
Vol. 41, No. I, January 1984 Printed in U.SA.
FERTILITY AND STERILITY Copyright" 1984 The American Fertility Society
Follicular fluid prostaglandins in endometriosis and ovarian hyperstimulation*
Olavi Ylikorkala, M.D.t Anssi Tenhunen, M.D. First Department of Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland
To study th~ presence of prostaglandin F2a (PGF2J, prostacyclin (PGI~, and thromboxane A2 (TxA~ in the human ovary, follicular fluid samples were collected with puncture at lapal'oscoPY in 17 women with pelvic endometriosis, 17 women with tubal occlusion and healthy ovaries, and 5 women with tubal occlusion and induced ovarian hyperstimulation between menstrual cycle days 8 and 18. The concentrations of the metabolites of PGF2a, PGI2, and TxA 2, i.e., 13,14-dihydro-15-keto PGF2a (M-PGF2J, 6-keto PGF1a, and TxB 2, respectively, were measured with radioimmunoassays. Each prostanoid was detected in follicular fluid, but their concentrations were unrelated to the menstrual cycle day at collection. Moreover, these concentrations were similar in various groups of patients. The data suggest that the human ovary produces PGF2a, PGI2, and TxA2 in vivo and that these prostanoids, as measured from follicular fluid, may not be of primary significance in ovulation or endometriosis. Fertil Steril41:66, 1984 :
I,
The follicular fluid (FF) bathes the granulosa cells and oocytes of the Graafian follicles. It contains high amounts of gonadotropins, prolactin, and steroids, which may be significant in determining the subsequent function of the corpus luteum and even the success rate of in vitro fertilization. 1 Also, prostaglandins (PGs), prostacyclin (PGI2), and thromboxane A2 (TxA2) are potent biologic mediators that are abundantly produced by various ovarian cells of human and animal origin in vitro. 2-7 Nevertheless, their role in oocyte maturation and ovulation has remained open in women, and they have never been measured from human follicular fluid. 7 The ovarian PGF2a,
PGI2, and TxA2 can be of special interest in endometriosis, because these patients have increased concentrations of the metabolites of PGF2a' PGI2, and TxA2 in their peritoneal fluid, according to most8 - 10 but not all reports. ll The present study was designed to determine the concentrations of 13,14-dihydro-15-keto PGF2a (MPGF2a, a metabolite of PGF2a), 6-keto PGF la (a breakdown product of PGI2), andthromboxane B2 (TxB 2, a breakdown product of TxA2) in the FF of women with healthy ovaries and in those with pelvic endometriosis. In addition, the effect of ovarian hyperstimulation on FF prostanoids was also studied.
Received May 2, 1983; revised and accepted July 20, 1983. *Supported by the Finnish Cancer Society and Yrjo Jahnsson Foundation. tReprint requests: Olavi Ylikorkala, M.D., First Department of Obstetrics and Gynecology, University of Helsinki, Haartmaninkatu 2, SF-00290 Helsinki 29, Finland.
66
Ylikorkala and Tenhunen Follicular fluid prostaglandins
MATERIALS AND METHODS PATIENTS
Thirty-nine women between 25 and 38 years of age were studied (Table 1). Thirty-four underFertility and Sterility
Table 1. Clinical Characteristics of the Study Populationsa Diagnosis
Endometriosis Tubal disorder Ovulation induction
No. of women
Age
at sampling
17 17 5
34.1 ± 0.8 29.0 ± 1.4 29.8 ± 2.1
12.4 ± 0.9 12.2 ± 1.1 13.2 ± 0.2
Cycle day
aMean ± standard error.
went laparoscopy between days 8 and 18 of the menstrual cycle as part of an evaluation for infertility. Twelve women proved to have mild endometriosis and 5 women had moderate endometriOSiS,12 whereas the other 17 women had unilateral or bilateral tubal occlusion but apparently healthy ovaries. The remaining five women also had tubal occlusion and volunteered for the in vitro fertilization program. Their ovaries were stimulated with 100 mg of clomiphene on cycle days 3 to 9, human menopausal gonadotropin (Humegon, Organon, Oss, The Netherlands), 75 IU on days 7 to 9, and human chorionic gonadotropin (Pregnyl, Organon), 10,000 IU on cycle day 12, for the growth of multiple follicles, which were aspirated on cycle day 13. No subject had used drugs known to interfere with the synthesis of PGs during the study cycle.
standard curves and the abilities of the two different-sized aliquots from the organic extracts to displace the tracers from antibodies confirmed the similarity of the compounds extracted from FF with authentic prostanoids. Due to the scarcity of FF samples, the recovery studies were carried out with samples of peritoneal fluid, the biochemical composition of which resembles that of FF.16 The recoveries of M-PGF 2"" 6-keto PGF 1"" and TxB2 added in peritoneal fluid were 77.4% ± 6.4%, 69.8% ± 4.6%, and 84.3% ± 7.8%, respectively (mean ± standard deviation, n = 10). STATISTICAL METHODS
The results were subjected to analysis of variance and t-testing according to the Bonferroni method. 17 Spearman's rank correlation was employed as well.
M~PGF2cx (pg/ml)
•2664 0 0
Vol. 41, No.1, January 1984
•
• • o
c
~c
8 100
••
•
•
600
0
400
CO
•.
i 0
• 8
0
• •
0
, i
OJ
I
0
I I 01348
6-keto-PGF1ot(pg/mllc
•
•
• •
I
800
MEASUREMENT OF PROSTAGLANDINS
The concentrations ofM-PGF2a (n = 26),6-keto PGF la (n = 39), and TxB2 (n = 39) in FF were measured with radioimmunoassays.13-l5 Briefly, a 0.5-ml sample was acidified with hydrochloric acid to pH 3.0 and extracted three times with a twofold excess of ethyl acetate. The combined organic phases were evaporated to dryness under nitrogen and dissolved in methanol. Two volumes from the methanol, both in duplicate, were taken for the radioimmunoassay of M-PGF 2a , 6-keto PGF la, and TxB2. The parallelism between the
0
•
o
SAMPLES
The Graafian follicle was aspirated gently at laparoscopy. In women with ovarian hyperstimulation, all the follicles (n = 4 to 7) were aspirated, but the fluid only from the largest ("leading") one was used in this study. The aspirates were transferred into heparinized tubes containing indomethacin (100 nmol), chilled at 4° C, and centrifuged. The supernates were stored at - 20° C until assayed. The samples visibly contaminated with blood were discarded.
o
8
• .e
0 0
• • •
oe
TxB2 (pg/mO 0
• • 8 200
0
&
C»
•
I
•
0
0
•, 8I
10
12
0 0
&e• I
14
•
• 5 I
16
•i 18
DA YS OF MENSTRUAL CYCLE Figure 1 The concentrations of M-PGF2a> PGF2a , and TxB2 in FF of patients with tubal occlusion (0), endometriosis (e), and induced ovulation (D). YlikorkaIa and Tenhunen Follicular fluid prostaglandins
67
Table 2. Concentrations (Mean ± Standard Error) of M-PGF2OJ 6-keto-PGF1a , and TxB2 in Follicular Fluid in Patients with Endometriosis, Tubal Disorders, and Induced Ovulationa Diagnosis
Endometriosis (n = 17) Tubal disorder (n = 17) Induced ovulation (n = 5)
M-PGFaa
6-keto-PGF1a
TxB2
pg/ml
pg/ml
pg/ml
759 ± (n = 562 ± (n = 385 ± (n =
215 431 ± 49 (n = 17) 10) 107 457 ± 69 (n = 17) 11) 37 505 ± 156 (n = 5) 5)
318 ± 32 (n = 17) 326 ± 38 (n = 17) 233 ± 37 (n = 5)
aNo difference is seen in these prostanoids among the groups.
RESULTS
All samples measured contained M-PGF2a (in a concentration range of 160 to 2664 pgiml), 6-keto PGF 10. (158 to 1346 pgiml), and TxB2 (101 to 678 pgiml). These concentrations were unrelated to the cycle day at collection (Fig. 1), diagnosis, or treatment of the patients (Table 2). DISCUSSION
The rises in the concentrations ofPGE and PGF in FF preceding ovulation in several species may suggest the implication of these prostanoids in ovum maturation and ovulation. 18 In addition to these classic PGs, the more recently found PGI2 and TxA2 may be of significance, because they are produced by the ovarian cells in vitro.6, 7 Being potently vasoactive compounds, 'PGI2 and TxA2 may change ovarian blood flow and vascular permeability, causing follicular rupture. 19 Furthermore, PGI2 stimulated cyclic adenosine monphosphate production by rat granulosa cells in vitro. 2o We present here evidence that human antral fluid contains M-PGF 2a , 6-keto PGF1a, and TxB2. Although it is impossible to deduce definitely which cells of the follicle produced these prostanoids, we regard the granulosa cells lining the follicle as a.most likely source because they generated PGs and PGI2 in rabbits21 , 22 and rats 6 in vitro. Also, the endothelial cells, which are abundant in ovarian vasculature and which normally release PGI2, may contribute to 6-keto PGF 10. in FF. The circulating blood is an unlikely origin of follicular prostanoids, since the concentrations of M-PGF2a , 6-keto-PGF1a , and TxB2 in peripheral blood, as measured by the same methods,13 were much lower than what was found here in FF. Whatever the origin of follicular prostanoids is, they showed no dependence on the cycle day and 68
were normal even in women with induced ovarian hyperstimulation. This is in contrast to luteinizing hormone- or human chorionic gonadotropininduced stimulation of PGE, PGF, and 6-keto PGF1a production by some animals in vivo or in vitro.6, 18 Our data do not seem to suggest a primary role for FF PGF2a , PGI2, and TxA2 in human ovum maturation and ovulation or in the development of the ovarian hyperstimulation syndrome in patients undergoing ovulation induction. This is in accordance with clinical experience that drugs capable of inhibiting PG synthesis do not prevent ovulation in women. 23 However, our findings must be interpreted with caution, because the puncture of the follicle may traumatically result in such a huge release of PGF20.' PGI2, and TxA2 that it masks the possible cyclicity of antral PGs. Endometriosis is often associated with infertility, even though the ovaries, tubes, and uterus are apparently healthy. The reason for the infertility of these patients can be a tuboovarian dysfunction, possibly caused by the smooth muscle cell activities of PGI2 and TxA2. Support for this speculation comes from increased concentrations of M-PGF 2a , 6-keto PGF 1a , and TxB2 in peritoneal fluid in endometriosis,8-10 although normal concentrations have also been reportedY We present here the first evidence that patients with endometriosis have normal concentrations of MPGF20.' 6-keto PGF la, and TxB2 in their antral fluid. Thus, the increased PG release in peritoneal fluid possibly associated with endometriosis does not derive from the follicle. Other sources like pelvic serosa24 and macrophages,25 should b~ considered as the origin of increased concentration ofPGs in the peritoneal fluid of patients with endometriosis. We conclude that the human FF contains MPGF20.' 6-keto PGF la, and TxB2. These concentrations are unrelated to the menstrual cycle day and are normal in patients with pelvic endometriosis and ovarian hyperstimulation.
Ylikorkala and Tenhunen Follicular fluid prostaglandins
Acknowledgment. The M-PGF2a antibody was a generous gift of Dr. John E. Pike, of The Upjohn Company, Kalamazoo,
Michigan.
REFERENCES 1. Carson RS, Trounson AO, Findlay JK: Successful fertili-
sation of human oocytes in vitro: concentration of estradiol-17J3, progesterone and androstenedione in the antral fluid of donor follicles. J Clin Endocrinol Metab 55:798, 1982
Fertility and Sterility
2. Shutt DA, Shearman RP, Lyneham RC, Clarke AH, McMahon GR, Goh P: Radioimmunoassay of progesterone, 17-hydroxyprogesterone, estradiol-17J3 and prostaglandin F in human corpus luteum. Steroids 26:299, 1975 3. Challis JRG, Calder AA, Dilley S, Foster CS, Hillier K, Hunter DJS, McKenzie IZ, Thorburn GD: Production of prostaglandins E and F2 by corpora lutea, corpora albicantes and stroma from the human ovary. J Endocrinol 68:401, 1976 4. Swanston lA, McNatty KP, Baird DT: Concentration of prostaglandin F 2n and steroids in human corpus luteum. J Endocrinol 73:115, 1977 5. Liedtke MP, Seifert B: Biosynthesis of prostaglandins in human ovarian tissue. Prostaglandins 16:825, 1978 6. Koos RD, Clark MR: Production of 6-keto-prostaglandin F 1n by rat granulosa cells in vitro. Endocrinology 111:1513, 1982 7. Poyser NL, Phillips CA: Gonadal function. In The Prostaglandin System, Endoperoxides, Prostacyclin and Thromboxanes, Edited by F Berti, GP Velo. New York, Plenum Press, 1981, p 301 8. Drake TS, O'Brien WF, Ramwell PW, Metz SA: Peritoneal fluid thromboxane B2 and 6-keto-prostaglandin F 1n in endometriosis. Am J Obstet Gynecol 140:401, 1981 9. Badawy SZA, Marshall L, Gabal AA, Nusbaum ML: The concentration of 13,14-dihydro-15-keto prostaglandin F 2n and prostaglandin E2 in peritoneal fluid of infertile patients with and without endometriosis. Fertil Steril 38:166, 1982 10. Ylikorkala 0, Koskimies A, Laatikainen T, Tenhunen A, Viinikka L: Peritoneal fluid 6-keto-prostaglandin F 1m thromboxane B2, estradiol and progesterone in endometriosis, tubal disorders and unexplained infertility. Obstet Gynecol. In press 11. Rock JA, Dubin NH, Ghodgaonkar RB, Bergquist CA, Erozan YS, Kimball AW Jr: Cul-de-sac fluid in women with endometriosis: fluid volume and prostanoid concentration during the proliferative phase of the cycle-days 8 to 12. Fertil Steril 37:747, 1982 12. Acosta A, Buttram V, Besch P, Malinak LR, Franklin RR, Vanderheyden JD: A proposed classification of pelvic endometriosis. Obstet Gynecol 42:19, 1973
Vol. 41, No.1, January 1984
13. Ylikorkala 0, Viinikka L: Prostacyclin, thromboxane and prostaglandin F 2n in maternal plasma during breastfeeding. Am J Obstet Gynecol 139:690, 1981 14. Ylikorkala 0, Kaila J, Viinikka L: Prostacyclin and thromboxane in diabetes. Br Med J 283:1148, 1981 15. Viinikka L, Ylikorkala 0: Measurement of thromboxane B2 in human plasma or serum using radioimmunoassay. Prostaglandins 20:559, 1980 16. Koninckx PR, Renaer M, Brosens IA: Origin of peritoneal fluid in women: an ovarian exudation product. Br J Obstet GynaecoI87:177, 1980 17. Wallenstein S, Zucker CL, Fleiss JL: Some statistical methods useful in circulation research. Circ Res 47:1, 1980 18. Poyser NL: Physiological role of prostaglandins in reproduction. In Prostaglandins and Thromboxanes, Edited by F Berti, B Samuelsson, GP Velo. New York, Plenum Press, 1977, p 205 19. Espey LL: Ovulation as an inflammatory reaction: a hypothesis. BioI Reprod 22:73, 1980 20. Goff AK, Zamecnik J, Ali M, Armstrong DT: Prostaglandin 12 stimulation of granulosa cell cyclic AMP production. Prostaglandins 15:875, 1978 21. Erickson GF, Challis JRG, Ryan KJ: Production of prostaglandin F by rabbit granulosa cell and theca tissue. J Reprod Fertil 49:133, 1977 22. Triebwasser WF, Clark RR, LeMaire WJ, Marsh JM: Localization and in vitro synthesis of prostaglandins in components of rabbit preovulatory Graafian follicles. Prostaglandins 16:621, 1978 23. Chaudhuri G, Elder MG: Lack of evidence for inhibition of ovulation by aspirin in women. Prostaglandins 11:727, 1976 24. Herman A, Claes M, Moncada S, Vane JR: Biosynthesis of PGI2 and 12-HETE by pericardium, pleura, peritoneum and aorta of the rabbit. Prostaglandins 18:439, 1979 25. Bray MA, Morley J: Prostaglandins and immunity. In The Prostaglandin System, Endoperoxides, Prostacyclin and Thromboxanes, Edited by F Berti, GP Velo. New York, Plenum Press, 1981, p 193
Ylikorkala and Tenhunen Follicular fluid prostaglandins
69
FERTILITY AND STERILITY Copyright" 1984 The American Fertility Society
Follicular fluid prostaglandins in endometriosis and ovarian hyperstimulation*
Olavi Ylikorkala, M.D.t Anssi Tenhunen, M.D. First Department of Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland
To study th~ presence of prostaglandin F2a (PGF2J, prostacyclin (PGI~, and thromboxane A2 (TxA~ in the human ovary, follicular fluid samples were collected with puncture at lapal'oscoPY in 17 women with pelvic endometriosis, 17 women with tubal occlusion and healthy ovaries, and 5 women with tubal occlusion and induced ovarian hyperstimulation between menstrual cycle days 8 and 18. The concentrations of the metabolites of PGF2a, PGI2, and TxA 2, i.e., 13,14-dihydro-15-keto PGF2a (M-PGF2J, 6-keto PGF1a, and TxB 2, respectively, were measured with radioimmunoassays. Each prostanoid was detected in follicular fluid, but their concentrations were unrelated to the menstrual cycle day at collection. Moreover, these concentrations were similar in various groups of patients. The data suggest that the human ovary produces PGF2a, PGI2, and TxA2 in vivo and that these prostanoids, as measured from follicular fluid, may not be of primary significance in ovulation or endometriosis. Fertil Steril41:66, 1984 :
I,
The follicular fluid (FF) bathes the granulosa cells and oocytes of the Graafian follicles. It contains high amounts of gonadotropins, prolactin, and steroids, which may be significant in determining the subsequent function of the corpus luteum and even the success rate of in vitro fertilization. 1 Also, prostaglandins (PGs), prostacyclin (PGI2), and thromboxane A2 (TxA2) are potent biologic mediators that are abundantly produced by various ovarian cells of human and animal origin in vitro. 2-7 Nevertheless, their role in oocyte maturation and ovulation has remained open in women, and they have never been measured from human follicular fluid. 7 The ovarian PGF2a,
PGI2, and TxA2 can be of special interest in endometriosis, because these patients have increased concentrations of the metabolites of PGF2a' PGI2, and TxA2 in their peritoneal fluid, according to most8 - 10 but not all reports. ll The present study was designed to determine the concentrations of 13,14-dihydro-15-keto PGF2a (MPGF2a, a metabolite of PGF2a), 6-keto PGF la (a breakdown product of PGI2), andthromboxane B2 (TxB 2, a breakdown product of TxA2) in the FF of women with healthy ovaries and in those with pelvic endometriosis. In addition, the effect of ovarian hyperstimulation on FF prostanoids was also studied.
Received May 2, 1983; revised and accepted July 20, 1983. *Supported by the Finnish Cancer Society and Yrjo Jahnsson Foundation. tReprint requests: Olavi Ylikorkala, M.D., First Department of Obstetrics and Gynecology, University of Helsinki, Haartmaninkatu 2, SF-00290 Helsinki 29, Finland.
66
Ylikorkala and Tenhunen Follicular fluid prostaglandins
MATERIALS AND METHODS PATIENTS
Thirty-nine women between 25 and 38 years of age were studied (Table 1). Thirty-four underFertility and Sterility
Table 1. Clinical Characteristics of the Study Populationsa Diagnosis
Endometriosis Tubal disorder Ovulation induction
No. of women
Age
at sampling
17 17 5
34.1 ± 0.8 29.0 ± 1.4 29.8 ± 2.1
12.4 ± 0.9 12.2 ± 1.1 13.2 ± 0.2
Cycle day
aMean ± standard error.
went laparoscopy between days 8 and 18 of the menstrual cycle as part of an evaluation for infertility. Twelve women proved to have mild endometriosis and 5 women had moderate endometriOSiS,12 whereas the other 17 women had unilateral or bilateral tubal occlusion but apparently healthy ovaries. The remaining five women also had tubal occlusion and volunteered for the in vitro fertilization program. Their ovaries were stimulated with 100 mg of clomiphene on cycle days 3 to 9, human menopausal gonadotropin (Humegon, Organon, Oss, The Netherlands), 75 IU on days 7 to 9, and human chorionic gonadotropin (Pregnyl, Organon), 10,000 IU on cycle day 12, for the growth of multiple follicles, which were aspirated on cycle day 13. No subject had used drugs known to interfere with the synthesis of PGs during the study cycle.
standard curves and the abilities of the two different-sized aliquots from the organic extracts to displace the tracers from antibodies confirmed the similarity of the compounds extracted from FF with authentic prostanoids. Due to the scarcity of FF samples, the recovery studies were carried out with samples of peritoneal fluid, the biochemical composition of which resembles that of FF.16 The recoveries of M-PGF 2"" 6-keto PGF 1"" and TxB2 added in peritoneal fluid were 77.4% ± 6.4%, 69.8% ± 4.6%, and 84.3% ± 7.8%, respectively (mean ± standard deviation, n = 10). STATISTICAL METHODS
The results were subjected to analysis of variance and t-testing according to the Bonferroni method. 17 Spearman's rank correlation was employed as well.
M~PGF2cx (pg/ml)
•2664 0 0
Vol. 41, No.1, January 1984
•
• • o
c
~c
8 100
••
•
•
600
0
400
CO
•.
i 0
• 8
0
• •
0
, i
OJ
I
0
I I 01348
6-keto-PGF1ot(pg/mllc
•
•
• •
I
800
MEASUREMENT OF PROSTAGLANDINS
The concentrations ofM-PGF2a (n = 26),6-keto PGF la (n = 39), and TxB2 (n = 39) in FF were measured with radioimmunoassays.13-l5 Briefly, a 0.5-ml sample was acidified with hydrochloric acid to pH 3.0 and extracted three times with a twofold excess of ethyl acetate. The combined organic phases were evaporated to dryness under nitrogen and dissolved in methanol. Two volumes from the methanol, both in duplicate, were taken for the radioimmunoassay of M-PGF 2a , 6-keto PGF la, and TxB2. The parallelism between the
0
•
o
SAMPLES
The Graafian follicle was aspirated gently at laparoscopy. In women with ovarian hyperstimulation, all the follicles (n = 4 to 7) were aspirated, but the fluid only from the largest ("leading") one was used in this study. The aspirates were transferred into heparinized tubes containing indomethacin (100 nmol), chilled at 4° C, and centrifuged. The supernates were stored at - 20° C until assayed. The samples visibly contaminated with blood were discarded.
o
8
• .e
0 0
• • •
oe
TxB2 (pg/mO 0
• • 8 200
0
&
C»
•
I
•
0
0
•, 8I
10
12
0 0
&e• I
14
•
• 5 I
16
•i 18
DA YS OF MENSTRUAL CYCLE Figure 1 The concentrations of M-PGF2a> PGF2a , and TxB2 in FF of patients with tubal occlusion (0), endometriosis (e), and induced ovulation (D). YlikorkaIa and Tenhunen Follicular fluid prostaglandins
67
Table 2. Concentrations (Mean ± Standard Error) of M-PGF2OJ 6-keto-PGF1a , and TxB2 in Follicular Fluid in Patients with Endometriosis, Tubal Disorders, and Induced Ovulationa Diagnosis
Endometriosis (n = 17) Tubal disorder (n = 17) Induced ovulation (n = 5)
M-PGFaa
6-keto-PGF1a
TxB2
pg/ml
pg/ml
pg/ml
759 ± (n = 562 ± (n = 385 ± (n =
215 431 ± 49 (n = 17) 10) 107 457 ± 69 (n = 17) 11) 37 505 ± 156 (n = 5) 5)
318 ± 32 (n = 17) 326 ± 38 (n = 17) 233 ± 37 (n = 5)
aNo difference is seen in these prostanoids among the groups.
RESULTS
All samples measured contained M-PGF2a (in a concentration range of 160 to 2664 pgiml), 6-keto PGF 10. (158 to 1346 pgiml), and TxB2 (101 to 678 pgiml). These concentrations were unrelated to the cycle day at collection (Fig. 1), diagnosis, or treatment of the patients (Table 2). DISCUSSION
The rises in the concentrations ofPGE and PGF in FF preceding ovulation in several species may suggest the implication of these prostanoids in ovum maturation and ovulation. 18 In addition to these classic PGs, the more recently found PGI2 and TxA2 may be of significance, because they are produced by the ovarian cells in vitro.6, 7 Being potently vasoactive compounds, 'PGI2 and TxA2 may change ovarian blood flow and vascular permeability, causing follicular rupture. 19 Furthermore, PGI2 stimulated cyclic adenosine monphosphate production by rat granulosa cells in vitro. 2o We present here evidence that human antral fluid contains M-PGF 2a , 6-keto PGF1a, and TxB2. Although it is impossible to deduce definitely which cells of the follicle produced these prostanoids, we regard the granulosa cells lining the follicle as a.most likely source because they generated PGs and PGI2 in rabbits21 , 22 and rats 6 in vitro. Also, the endothelial cells, which are abundant in ovarian vasculature and which normally release PGI2, may contribute to 6-keto PGF 10. in FF. The circulating blood is an unlikely origin of follicular prostanoids, since the concentrations of M-PGF2a , 6-keto-PGF1a , and TxB2 in peripheral blood, as measured by the same methods,13 were much lower than what was found here in FF. Whatever the origin of follicular prostanoids is, they showed no dependence on the cycle day and 68
were normal even in women with induced ovarian hyperstimulation. This is in contrast to luteinizing hormone- or human chorionic gonadotropininduced stimulation of PGE, PGF, and 6-keto PGF1a production by some animals in vivo or in vitro.6, 18 Our data do not seem to suggest a primary role for FF PGF2a , PGI2, and TxA2 in human ovum maturation and ovulation or in the development of the ovarian hyperstimulation syndrome in patients undergoing ovulation induction. This is in accordance with clinical experience that drugs capable of inhibiting PG synthesis do not prevent ovulation in women. 23 However, our findings must be interpreted with caution, because the puncture of the follicle may traumatically result in such a huge release of PGF20.' PGI2, and TxA2 that it masks the possible cyclicity of antral PGs. Endometriosis is often associated with infertility, even though the ovaries, tubes, and uterus are apparently healthy. The reason for the infertility of these patients can be a tuboovarian dysfunction, possibly caused by the smooth muscle cell activities of PGI2 and TxA2. Support for this speculation comes from increased concentrations of M-PGF 2a , 6-keto PGF 1a , and TxB2 in peritoneal fluid in endometriosis,8-10 although normal concentrations have also been reportedY We present here the first evidence that patients with endometriosis have normal concentrations of MPGF20.' 6-keto PGF la, and TxB2 in their antral fluid. Thus, the increased PG release in peritoneal fluid possibly associated with endometriosis does not derive from the follicle. Other sources like pelvic serosa24 and macrophages,25 should b~ considered as the origin of increased concentration ofPGs in the peritoneal fluid of patients with endometriosis. We conclude that the human FF contains MPGF20.' 6-keto PGF la, and TxB2. These concentrations are unrelated to the menstrual cycle day and are normal in patients with pelvic endometriosis and ovarian hyperstimulation.
Ylikorkala and Tenhunen Follicular fluid prostaglandins
Acknowledgment. The M-PGF2a antibody was a generous gift of Dr. John E. Pike, of The Upjohn Company, Kalamazoo,
Michigan.
REFERENCES 1. Carson RS, Trounson AO, Findlay JK: Successful fertili-
sation of human oocytes in vitro: concentration of estradiol-17J3, progesterone and androstenedione in the antral fluid of donor follicles. J Clin Endocrinol Metab 55:798, 1982
Fertility and Sterility
2. Shutt DA, Shearman RP, Lyneham RC, Clarke AH, McMahon GR, Goh P: Radioimmunoassay of progesterone, 17-hydroxyprogesterone, estradiol-17J3 and prostaglandin F in human corpus luteum. Steroids 26:299, 1975 3. Challis JRG, Calder AA, Dilley S, Foster CS, Hillier K, Hunter DJS, McKenzie IZ, Thorburn GD: Production of prostaglandins E and F2 by corpora lutea, corpora albicantes and stroma from the human ovary. J Endocrinol 68:401, 1976 4. Swanston lA, McNatty KP, Baird DT: Concentration of prostaglandin F 2n and steroids in human corpus luteum. J Endocrinol 73:115, 1977 5. Liedtke MP, Seifert B: Biosynthesis of prostaglandins in human ovarian tissue. Prostaglandins 16:825, 1978 6. Koos RD, Clark MR: Production of 6-keto-prostaglandin F 1n by rat granulosa cells in vitro. Endocrinology 111:1513, 1982 7. Poyser NL, Phillips CA: Gonadal function. In The Prostaglandin System, Endoperoxides, Prostacyclin and Thromboxanes, Edited by F Berti, GP Velo. New York, Plenum Press, 1981, p 301 8. Drake TS, O'Brien WF, Ramwell PW, Metz SA: Peritoneal fluid thromboxane B2 and 6-keto-prostaglandin F 1n in endometriosis. Am J Obstet Gynecol 140:401, 1981 9. Badawy SZA, Marshall L, Gabal AA, Nusbaum ML: The concentration of 13,14-dihydro-15-keto prostaglandin F 2n and prostaglandin E2 in peritoneal fluid of infertile patients with and without endometriosis. Fertil Steril 38:166, 1982 10. Ylikorkala 0, Koskimies A, Laatikainen T, Tenhunen A, Viinikka L: Peritoneal fluid 6-keto-prostaglandin F 1m thromboxane B2, estradiol and progesterone in endometriosis, tubal disorders and unexplained infertility. Obstet Gynecol. In press 11. Rock JA, Dubin NH, Ghodgaonkar RB, Bergquist CA, Erozan YS, Kimball AW Jr: Cul-de-sac fluid in women with endometriosis: fluid volume and prostanoid concentration during the proliferative phase of the cycle-days 8 to 12. Fertil Steril 37:747, 1982 12. Acosta A, Buttram V, Besch P, Malinak LR, Franklin RR, Vanderheyden JD: A proposed classification of pelvic endometriosis. Obstet Gynecol 42:19, 1973
Vol. 41, No.1, January 1984
13. Ylikorkala 0, Viinikka L: Prostacyclin, thromboxane and prostaglandin F 2n in maternal plasma during breastfeeding. Am J Obstet Gynecol 139:690, 1981 14. Ylikorkala 0, Kaila J, Viinikka L: Prostacyclin and thromboxane in diabetes. Br Med J 283:1148, 1981 15. Viinikka L, Ylikorkala 0: Measurement of thromboxane B2 in human plasma or serum using radioimmunoassay. Prostaglandins 20:559, 1980 16. Koninckx PR, Renaer M, Brosens IA: Origin of peritoneal fluid in women: an ovarian exudation product. Br J Obstet GynaecoI87:177, 1980 17. Wallenstein S, Zucker CL, Fleiss JL: Some statistical methods useful in circulation research. Circ Res 47:1, 1980 18. Poyser NL: Physiological role of prostaglandins in reproduction. In Prostaglandins and Thromboxanes, Edited by F Berti, B Samuelsson, GP Velo. New York, Plenum Press, 1977, p 205 19. Espey LL: Ovulation as an inflammatory reaction: a hypothesis. BioI Reprod 22:73, 1980 20. Goff AK, Zamecnik J, Ali M, Armstrong DT: Prostaglandin 12 stimulation of granulosa cell cyclic AMP production. Prostaglandins 15:875, 1978 21. Erickson GF, Challis JRG, Ryan KJ: Production of prostaglandin F by rabbit granulosa cell and theca tissue. J Reprod Fertil 49:133, 1977 22. Triebwasser WF, Clark RR, LeMaire WJ, Marsh JM: Localization and in vitro synthesis of prostaglandins in components of rabbit preovulatory Graafian follicles. Prostaglandins 16:621, 1978 23. Chaudhuri G, Elder MG: Lack of evidence for inhibition of ovulation by aspirin in women. Prostaglandins 11:727, 1976 24. Herman A, Claes M, Moncada S, Vane JR: Biosynthesis of PGI2 and 12-HETE by pericardium, pleura, peritoneum and aorta of the rabbit. Prostaglandins 18:439, 1979 25. Bray MA, Morley J: Prostaglandins and immunity. In The Prostaglandin System, Endoperoxides, Prostacyclin and Thromboxanes, Edited by F Berti, GP Velo. New York, Plenum Press, 1981, p 193
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