A factor inhibiting ovum capture by the oviductal fimbriae present in endometriosis peritoneal fluid

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1 FERTILITY AND STERILITY Copyright ~ 1986 The American Fertility Society Vol. 46, No.6, December 1986 Printed in U.SA. A factor inhibiting ovum capture by the oviductal fimbriae present in endometriosis peritoneal fluid Hiroshi Suginami, M.D. * Kohji Yano, M.D. Katsuichi Watanabe, M.D. Shumpei Matsuura, M.D. Department of Obstetrics and Gynecology, School of Medicine, Ehime University, Shigenobu, Ehime, Japan The cumulus-fimbria interaction was investigated in vitro with endometriosis peritoneal fluid (PF). On the oviductal fimbria of the golden hamster, incubated with endometriosis and nonendometriosis PF and culture medium 199 contained 4% bovine serum albumin (control), was placed the mouse oocyte-cumulus complex at 5-minute intervals for 60 to 70 minutes. Incubation periods until the loss of fimbrial capability of ovum capture (ovum capturability disappearance time [OCDT]) were 23.3 ± 2.7 (standard error of the mean), 51.7 ± 2.9, and 61.3 ± 0.9 minutes with endometriosis, nonendometriosis PF, and control, respectively. OCnT was significantly decreased with cell-free and ultrafiltrated endometriosis PF containing a molecular size> 100,000 (26.0 ± 2.8 and 26.1 ± 2.5 minutes, respectively). A factor inhibiting fimbrial capability of ovum capture is present in endometriosis PF. Fertil Steril 46:1140, 1986 Endometriosis is a disease affecting women of reproductive age and causes infertility. Although it is not rare and has been extensively investigated, its cause and the genesis of symptoms and infertility associated with the disease are still controversial. Various mechanisms, such as interference with sexual function, ovulation, ovum transport, fertilization, and implantation, have been postulated as the cause of infertility associated with endometriosis. The frequent occurrence of the luteinized unruptured follicle syndrome was documented in women with endometrio- Received March 10, 1986; revised and accepted August 11, *Reprint requests: Hiroshi Suginami, M.D., Department of Obstetrics and Gynecology, School of Medicine, Ehime University, Shigenobu, Ehime , Japan. sis,1, 2 whereas no difference was later pointed out in the distribution of this finding in women with and without. endometriosis. 3 Increased peritoneal concentration of prostaglandins may be instrumental in altering tubal and uterine motility and in preventing normal ovum transport from the ovary to the uterus, although there are diametrically adverse reports in terms of intraperitoneal prostaglandin concentrations in women with endometriosis.4-10 Enhanced intraperitoneal and intratubal macrophage phagocytosis of spermatozoa was demonstrated in vitro in endometriosis,11, 12 although its in vivo contribution is not yet proven and there is a contrary reportp Alteration in the nature of oocytes was suggested by the reduced in vitro fertilizability of oocytes retrieved from women with endometriosis.14 Thus none of them is conclusive as to the nature of infertility associated with endometriosis Suginami et al. Ovum capture inhibitor in endometriosis Fertility and Sterility

2 Although the cause of endometriosis-associated infertility is unknown, a variety of treatments have been performed, i.e., conservative and laparoscopic surgery, pseudopregnancy, pseudomenopause, and expectant management after laparoscopic diagnosis, which, regardless of the method employed, has been associated with an increased rate of conception. 15 Among them, the result of expectant management, especially in mild endometriosis, is impressive. 16, 17 An increased pregnancy rate in patients with mild endometriosis and many years of infertility suggests that a component of the diagnostic procedure employed could act in a therapeutic manner. Laparoscopic manipulation, aspiration of peritoneal fluid (PF), dilation of cervical canal, and/or tubal lavage could be the candidates, although this is not yet proven. This study was conducted to elucidate the mechanisms of endometriosis-associated infertility with a working hypothesis that a factor inhibiting ovum capture and transfer by the oviductal fimbria might be present in PF of patients with endometriosis. ANIMALS MATERIALS AND METHODS Adult female Golden hamsters aged 7 to 8 weeks were used as the source of oviducts and oviductal fimbriae. They were kept under controlled lighting (light on 7:00 A.M. to 7:00 P.M.) with laboratory chow and water ad libitum. No attention was paid to the cycle date of the Golden hamsters, because no cyclic difference was reported in the ability of oviductal fimbria to capture and transfer ovulatory cumulus oophorus. 18 Immature female ICR mice aged 4 weeks were superovulated with an intraperitoneal bolus of 5 IU of pregnant mare serum gonadotropin and a subsequent intraperitoneal bolus of 5 IU of human chorionic gonadotropin at hours. The mice were sacrificed 16 hours after the hcg injection, and the oocyte-cumulus complexes in the oviducts were collected. The oocyte-cumulus complex was stained with 0.015% methylene blue in Dulbecco's phosphate buffered saline (GIBCO Laboratories, Chagrin Falls, OH) for the purpose of easy observation, rinsed and immersed in tissue culture Medium 199 (TC-199; GIBCO). In the current study, different species of animals were used for investigation of cumulusfimbria interaction. This was because the oviductal fimbria of golden hamster was reported to capture readily the oocyte-cumulus complexes originating from different animal species, e.g., mouse, rat, and rabbit, besides its own 18 and for the sake of economy. The oviducts and the oocytecumulus complexes were subjected to the experiments immediately and within 5 hours after preparation, respectively. PERITONEAL FLUID Fifteen and 9 patients with and without endometriosis, respectively, participated in the current study. All gave informed consent. The profiles of the patients are listed in Tables 1 and 2. PF was aspirated laparoscopically from the anterior uterovesical space and/or posterior cul-de-sac of the patients with endometriosis and other diseases, under direct vision, prior to laparoscopic manipulation. The PF was aspirated by the same person for all patients. All of the PF specimens aspirated from endometriosis patients and some of the PF specimens aspirated from nonendometriosis patients were contaminated with blood, although the extent of blood contamination differed (Tables 1 and 2). Blood contamination was not attributable to the technique of laparoscopy. The PF specimens were either subjected to experiment I without pretreatment or stored at - 70 C in aliquots after removal of cellular components by centrifugation at 3000 rpm for 15 minutes at room temperature for experiments II and III. TC-199 containing 4% bovine serum albumin (Fraction V; Sigma Chemical Co., St. Louis, MO) served as the control. EXPERIMENT I The Golden hamster oviduct was placed in a chamber of a Nunclon 4-Well-Multidish (Nunc AlS, Roskilde, Denmark) and was incubated at 37 C with 1 to 2 ml of native PF specimens from endometriosis or nonendometriosispatients and the contralateral oviduct with the same amount of control medium. On the oviductal fimbria was placed the oocyte-cumulus complex, with the aid of a micropipette. The capture and transfer of the oocyte-cumulus complex by the oviductal fimbria was investigated with the aid of a dissecting microscope model SZH-141 (Olympus Corp., Tokyo, Japan). The above procedures were repeated at 5-minute intervals for 60 to 70 minutes. The incubation period from the initiation of the experi- Vol. 46, No.6, December 1986 Suginami et al. Ovum capture inhibitor in endometriosis 1141

3 Table 1. Profiles of Patients With Endometriosis Patient Age Gravida/para Infertile L-scope period day" Stage b Amount PF Hematocrit Remarks yrs yrs ml % /0 7 3 III /0 6 4 II /0 6 7 IV /0 2 7 I Pregnant during expectant management / I II II 30 < / II / II / II Pregnant during expectant management /0 4 II 80 < 0.2 Pregnant during expectant management /0 11 I 45 < 0.2 Spouse: varicocele /0 3 IV / II / II 18 < 0.2 "The cycle date when laparoscopy was performed. brevised American Fertility Society Classification of Endometriosis 25 was employed. ment until the ability of the oviductal fimb:fia to capture the ovum disappeared was designated the ovum capturability disappearance time (OCDT). In cases where the fimbrial capability of ovum capture was preserved beyond the experimental period, the OCDT was regarded as 60 or 70 minutes. The OCDTs were determined for the PF specimens and controls. EXPERIMENT II For assessment.,of the influence by the cellular components included in endometriosis, and nonendometriosis PF, the PF specimens were centrif\lged at 3000 rpm for 15 minutes at room temperature. The OCDTs were determined with the supernatants of centrifuged PF specimens. EXPERIMENT III For the preliminary estimation of the molecular size of the factor influencing the fimbriacumulus interaction, supernatants of the centrifuged PF specimens were ultrafiltrated with the aid of the Millipore SJGC and SJHK ultrafiltration kits (Millipore Corp., Bedford, MA), admitting substances having molecular sizes of < 10,000 and < 100,000 to be passed through, respectively. One milliliter of the centrifuged supernatant of endometriosis and nonendometriosis PF were transferred into the upper chaniber of the SJHK and air pressure was applied at 4 C according to the manual included in the kit. The SJHK filtrate was then transferred to the upper chamber of the SJGC and treated similarly. The residues remaining in the upper chambers of the SJHK and SJGC, i.e., substances having molecular sizes of> 100,000 and from 10,000 to 100,000, respectively, were dissolved in 1 ml of TC-199. The OCDTs were determined with the fractions of uitrafiltrated endometriosis and nonendometriosis PF, i.e., the SJGC filtrates (containing substances having molecular sizes of < 10,000), the SJGC residues, and the SJHK residues. STATISTICAL ANALYSIS The OCDTs obtained were analyzed statistically by one-way analysis of variance and Student's t-test. EXPERIMENT I RESULTS The mouse ovum surrounded by expanded cumulus oophorus was readily picked up by the oviductal fimbria of golden hamster and was transferred to the oviductal ampulla within seconds throughout the whole period of experiment, when the oviduct was incubated with the control medium regardless of cycle date of the animal (Fig. 1). The OCDT was 61.3 ± 0.9 (standard error of 1142 Suginami et al. Ovum capture inhibitor in endometriosis Fertility and Sterility

4 Table 2. Profile of Patients Without Endometriosis Patient Age Gravida/para L-scope daya 3/2 4/2 3/3 0/ /0 0/0 0/0 11 Laparoscopic diagnosis yrs Uterine myomata Uterine myomata Uterine myomata Polycystic ovary Polycystic ovary Polycystic ovary Tubal obstruction Tubal obstruction Rudimentary uterus NEe NE NE PF Amount Hematocrit b ml % < 0.2 athe cycle date when laparoscopic examination was performed. bthe sample specimens were not contaminated with blood when indicated with ( - ). ene, not estimated due to a long period of anovulation. the mean) minutes (n = ) with the control medium (Table 3). Fimbrial ovum capture was ready during the early period of experiment with endometriosis and non endometriosis PF specimens, as observed with the control medium. The velocity of transfer was, however, blunted as the experiment proceeded with the endometriosis PF and some of the nonendometriosis PF specimens, and finally the fimbrial capability to capture the ovum disappeared. The OCDT was 51.7 ± 2.9 (n = 9) minutes with nonendometriosis PF specimens, being slightly but significantly shorter than that with control medium (P < 0.01). However, the OCDT with endometriosis PF was 23.3 ± 2.7 minutes (n = 15) (Table 3), which was significantly shorter than those with control medium and with nonendometriosis PF (P < 0.001). The oviducts exhibited peristalsis even after the fimbrial capability to capture the ovum was lost. The capability was partially resumed by extensive rinsing of the oviducts with TC-199. EXPERIMENT III The OCDTs were 62.2 ± 1.5 (n = 9),62.8 ± 1.5 (n = 9), and 26.1 ± 2.5 (n = 9) minutes with SJGC filtrates, the SJGC residues, and the SJHK residues of endometriosis PF, respectively. The corresponding OCDTs with nonendometriosis PF were 63.1 ± 1.6 (n = 8), 60.6 ± 2.0 (n = 8), and 58.1 ± 2.1 (n = 8) minutes, respectively (Table 3). The OCDT with the SJHK residues of endome- EXPERIMENT II The OCDTs were 26.0 ± 2.8 (n = 10) and 53.8 ± 2.6 (n = 8) minutes with the supernatants of centrifuged endometriosis and nonendometriosis PF, respectively (Table 3). These values were not different from those with their corresponding native PF specimens. A significant difference was observed between the OCDT with cell-free endometriosis PF and those with cell-free nonendometriosis PF and the control medium (P < 0.001). Vol. 46, No.6, December 1986 Figure 1 An ovum surrounded by expanded cumulus oophorus was placed on the oviductal fimbria (A). The cumulus was captured by the fimbria (B) and transferred toward the oviductal ampulla (C). The cumulus was finally trapped in the oviduct (D). The capture and transfer of the cumulus oophorus by the oviductal fimbria was completed within seconds during the early period of experiment with endometriosis PF and throughout the experiment with TC-199. The ability of the fimbria to capture the cumulus-oophorus was lost as the experiment proceeded with endometriosis PF. Suginami et a1. Ovum capture inhibitor in endometriosis 1143

5 T Table 3_ oent with Native and Pretreated PF Aspirated Laparoscopically from Patients With and Without Endometriosis a Endometriosis PF Nonendometriosis PF TC-199 with 4% bovine serum albumin Native 23.3 ± 2.7 (15)b 51.7 ± 2.9 (9) 61.3 ± 0.9 () Cell-free 26.0 ± 2.8 (lo)b 53.8 ± 2.6 (8) Ultrafiltration < 10,000 10, ,000 > 100, ± 1.5 (9) 63.1 ± 1.6 (8) 62.2 ± 1.5 (9) 60.6 ± 2.0 (8) anumbers in parentheses denote number of experiments. bstatistical significance (P < 0.001) versus the corresponding values with nonendometriosis PF and control ± 2.5 (9)b 58.1 ± 2.1 (8) triosis PF exhibited statistical difference from those with the remaining fractions of ultrafiltrated endometriosis PF and those with all of the ultrafiltrated fractions of nonendometriosis PF (P < 0.001). DISCUSSION Endometriosis affects women of reproductive age and causes infertility. It can be diagnosed with the use of laparoscopy in 15% to 25% of infertile women, and in patients with otherwise unexplained infertility the frequency of endometriosis may be as high as 70% to 80%.19 Although endometriosis is not rare, its cause and the cause of infertility in patients bearing this disorder are unknown. Various possible mechanisms, such as interference with sexual function, ovulation, ovum transport, fertilization, and implantation have been postulated and demonstrated to occur in some, but not all, patients with endometriosis. 15 It seems reasonable to attribute endometriosisassociated infertility to the mechanical disturbance of ovum capture by the oviductal fimbriae due to adhesion and/or anatomic dislocation of the female genital organs, because endometriosis causes adhesion or ovarian enlargement in advanced cases. However, the association of endometriosis with infertility has been reported to be evident even in the absence of the damage to the oviducts and ovaries, which is in agreement with our experience. It is not easy to identify the mechanical factor by laparoscopic examination when it is performed on women with cases of minimal or mild endometriosis who are still infertile. A cellular component in endometriosis PF was raised as a factor causing sterility in endometriosis. It was reported that in patients with endometriosis the number of macrophages was more increased in their PF than in PF of normal fertile women and of infertile women without endometriosis and sperm phagocytosis of macrophages was rapacious.1l Furthermore, the number of oviductal macrophages has been documented to be significantly higher in patients with endometriosis who underwent conservative surgery than in either normal fertile women or in infertile women with tubal obstruction and appeared to be related with the number of peritoneal macrophages.12 These authors claimed that increased macrophages and their enhanced sperm phagocytosis could disturb ovum fertilization and cause infertility. Macrophages could inhibit fertilization by mediating damage to oocytes21 or zygotes.22 However, our understanding of the data presented by Muscato et al.1l is that sperm phagocytosis of macrophages was still rather accentuated in fertile women who underwent laparoscopic tubal cauterization, compared with that in normal women who underwent tubal recanalization. It seems natural that the number of intraperitoneal macrophages is more increased in women with tubal patency than in their counterparts, because a sperm is a foreign body in the female genital tract and in the peritoneal cavity, and a macrophage has a duty to trap and digest a foreign body.23, In addition, it was reported that phagocytic capacity of macrophages in PF of endometriosis patients did not differ from that in normal women.13 Thus the role of macro phages in endometriosis-associated infertility remains open for discussion. Another possible explanation for endometriosis-associated infertility is that increased intraperitoneal levels of prostaglandins and prostanoids cause luteolysis or enhanced tubal contractility or both. However, the role of these agents in the genesis of infertility in endometriosis is still controversial. There are diametrically adverse reports oflevels of these agents in the endometriosis PF.4-10 The enormous difference reported in the levels of these agents might be attributable both to the timing and the methods of aspiration 1144 Suginami et al. Ovum capture inhibitor in endometriosis Fertility and Sterility

6 of PF and to the quality control of the assay systems. Prostaglandins and prostanoids are labile and easily metabolized to biologically inactive forms, and there is no evidence showing disturbed in situ oviductal movement at the moment of ovulation by these agents. Furthermore, increased prostaglandin and prostanoid levels were reported not only in endometriosis PF but also in PF obtained from patients without endometriosis. 9, 10 With these results it is difficult to draw a definite conclusion on the role of these agents in infertility associated with endometriosis. Recently, Mahi-Brown and Yanagimachi clearly demonstrated the interaction between ovulatory cumulus oophorus and the oviductal fimbria. 18 The oviductal fimbria readily picks up the ovulatory oocyte-cumulus complex but not the immature one or the cumulus-free oocyte. Furthermore, the fimbria picks up loose connective tissues but not dense connective tissues. The characteristic features of the materials being picked up by the oviductal fimbria is the expanded intercellular matrix, where proteoglycans are abundant. The authors claimed that the oviductal fimbria recognizes intercellular matrix as the first step of ovum capture and transfer. In light of this cumulus-fimbria interaction, we conducted the current study with a working hypothesis that there might be a substance inhibiting the cumulus-fimbria interaction in endometriosis PF, which might cause the failure in ovum capture by the oviductal fimbria, resulting in infertility. Our experiments consisted of microscopic investigation of the changes in the capability of the fimbria to capture and transport the oocyte-cumulus complex when incubated with native and pretreated PF aspirated from patients with and without endometriosis. The oviductal fimbriae were randomly obtained from adult female Golden hamsters at various cycle dates. As was analogized from the homogeneity in OCDTs with the control medium, there was no cyclic variation in the fimbrial capability to capture the ovulatory oocyte-cumulus complex. Although the oocyte-cumulus complex originating from the mouse, instead of the golden hamster, was used in the experiments for the sake of economy, the experiments were successful with the vigorous activity of the golden hamster fimbria, as was observed by Mahi-Brown and Yanagimachi. 18 This study clearly demonstrates the presence of a factor inhibiting ovum capture by the oviductal fimbria in vitro (ovum capture inhibitor [OCID in the PF aspirated from patients with endometriosis. The OCI is water-soluble and has a molecular size> 100,000. Although the two groups of patients who participated in the study were not fully matched, the data obtained were quite conclusive. This is only a preliminary report; we need more concrete evidence for the origin of OCI and its in vivo contribution in infertility associated with endometriosis. The identification of OCI would provide us with new concepts as to the cause of endometriosis-associated infertility and in the management of infertile patients with endometriosis. Further qualitative and quantitative analyses of the OCI are in progress. REFERENCES 1. Koninckx PR, Heyns WJ, Corvelyn PA, Brosens IA: Delayed onset ofluteinization as a cause of infertility. Fertil Steril 29:266, Marik J, Hulka J: Luteinized unruptured follicle syndrome: a subtle cause of infertility. Fertil Steril 29:270, Dmowski WP, Rao R, Scommegna A: The luteinized unruptured follicle syndrome and endometriosis. Fertil Steril 33:30, Drake TS, O'Brien WF, Ramwell PW, Metz SA: Peritoneal fluid thromboxane B2 and 6-keto-prostaglandin FIn in endometriosis. 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