American Journal of Obstetrics and Gynecology
Volume 174 • Number 6 • June 1996
Copyright © 1996 Mosby-Year Book, Inc.

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TRANSACTIONS OF THE SIXTY-SECOND ANNUAL MEETING OF THE PACIFIC COAST OBSTETRICAL AND GYNECOLOGICAL SOCIETY

Dr.Sinan DOĞANTÜRK

Ankara

The effect of peritoneal fluid from patients with endometriosis on human sperm function in vitro

 

Roy T. Nakayama MD

Honolulu, Hawaii

Department of Obstetrics and Gynecology,
John A. Burns School of Medicine, University of Hawaii.

OBJECTIVE: Our purpose was to evaluate the effect of peritoneal fluid from women with endometriosis on sperm motility and function in an in vitro model.

STUDY DESIGN: Peritoneal fluid was collected at laparoscopy from patients with and without endometriosis. Human donor sperm was diluted with this fluid, and its effect on sperm function and motility was measured with the zona-free hamster egg sperm penetration assay and computer-assisted semen analysis.

RESULTS: The mean number of eggs penetrated by the sperm mixed with peritoneal fluid from patients with endometriosis was significantly fewer than the number penetrated by the sperm mixed with fluid from control patients (22.9 ± 5.31 vs 44.4 ± 4.96, p < 0.01, Student t test, n = 20). When evaluated by computer-assisted semen analysis, sperm mixed with peritoneal fluid from patients with endometriosis showed a significant decrease in mean swimming velocity compared with sperm mixed with peritoneal fluid from control patients (54.0 ± 1.77 vs 59.2 ± 1.05, p = 0.02, Student t test, n = 20). A significant increase in the fraction of sperm swimming at slower velocities was also found. A trend toward a positive correlation between eggs penetrated and sperm velocity was seen, but statistical significance was not achieved (correlation coefficient 0.4392, p = 0.053, n = 20).

CONCLUSION: These data suggest that substances found in the peritoneal fluid of patients with endometriosis could contribute to infertility through impairment of both sperm function and motion kinematics. (Am J Obstet Gynecol 1996;174:1779-85.)

Key words:

Sperm motility and function

endometriosis

peritoneal fluid

computer-assisted semen analysis

hamster egg penetration assay

Supported in part by Kapiolani Medical Center for Women and Children.

Presented at the Sixty-second Annual Meeting of the Pacific Coast Obstetrical and Gynecological Society, Squaw Valley, California, September 16-21, 1995.

Reprint requests: Tod C. Aeby, MD, University of Hawaii, John A. Burns School of Medicine, Department of Obstetrics and Gynecology, Kapiolani Medical Center for Women and Children, 1319 Punahou St., Honolulu, HI 96826.

Copyright © 1996 by Mosby -Year Book, Inc.

6/6/72188

Minimal endometriosis is commonly diagnosed among women undergoing evaluation for infertility and, although controversial, is thought to contribute to the couple's inability to become pregnant. It is speculated that a substance or substances from peritoneal implants enter the peritoneal fluid, interfering with the reproductive process. Comparing peritoneal fluid taken from patients with endometriosis with peritoneal fluid from those without endometriosis, investigators have shown an increased volume in the luteal phase ^{[1] [2]} ; increased concentrations of prostaglandins, ^{[2] [3]} interleukin-1, ^[4] tumor necrosis factor, ^[5] and transforming growth factor-beta ^[6] ; altered progesterone, protease inhibitor, and protein levels ^[7] ; decreased platelet-activating factor acetylhydrolate activity ^[8] ; and an increased number of activated macrophages. ^{[2] [9]}

Peritoneal fluid diffusing into the tubal and endometrial environment has an opportunity to affect sperm and their interaction with the oocyte. Previous studies of the effect of endometriosis on sperm motility have reached conflicting conclusions. Comparing peritoneal fluid from women with and without endometriosis, Muscato et al. ^[10] were able to demonstrate increased sperm phagocytosis by macrophages in patients with the disease. Stone and Himsl, ^[11] however, could demonstrate no difference in the recovery of motile sperm from the peritoneal cavity of patients with and without endometriosis undergoing laparoscopy immediately after artificial insemination. Curtis et al., ^[12] by use of centrifuged, untreated fluids with unwashed sperm, and Burke, ^[13] by use of filtered and heat-treated fluids, were able to demonstrate reductions in several motility parameters measured by various computer-assisted semen analyses. On the other hand, Bielfeld et al. ^[14] with untreated pooled fluids and a straw swim-up technique and Halme and Hall ^[15] with centrifuged, heat-treated fluids failed to find any difference in sperm mo-tility.

In reviewing studies that used oocyte penetration assays, Chacho et al. ^[2] demonstrated that sperm mixed with

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uncentrifuged peritoneal fluid from women with endometriosis penetrated significantly fewer zona-free hamster eggs, compared with sperm mixed with fluid from controls. Conversely, Halme and Hall ^[15] with heat-treated centrifuged fluid found no difference in the number of zona-free hamster eggs penetrated. Sueldo et al., ^[16] working with a murine sperm-oocyte model, showed a significant reduction in oocyte penetration with centrifuged peritoneal fluid from women with endometriosis, but this effect was lost when the fluid was heat inactivated.

Computer-assisted semen analysis has been shown to be a sensitive measure of sperm motility and general vigor, ^[17] whereas the zona-free hamster egg sperm penetration assay has been shown to be a valid test for subtle deficiencies in sperm fertilization capacity. ^[18] The purpose of this study was to use these two modalities, controlling for previously identified confounding variables, to further assess the effect of minimal endometriosis on sperm function and motility in an in vitro model.

Material and methods

This study was reviewed and approved by the Institutional Review Committee of Kapiolani Medical Center for Women and Children.

Peritoneal fluid was collected from 20 women undergoing laparoscopy for various indications. Ten of these women had an otherwise normal pelvis but met the American Fertility Society criteria for minimal (stage I) endometriosis. ^[19] Ten women with normal laparoscopic findings served as controls. The fluid was collected from the posterior cul-de-sac and transported on ice to the laboratory where it were centrifuged at 600 g for 5 minutes. The supernate was then frozen and stored at -20° C. All laparoscopies were performed by a single investigator, and subsequent tests were done by a second investigator blinded to the laparoscopic findings. The samples were batched and analyzed on a single day under identical conditions.

Zona-free hamster egg penetration assays were performed as described by Rogers et al. ^[20] with human sperm from a single donor. After liquefaction, semen was layered onto discontinuous Percoll gradients (Pharmacia, Uppsala) of 90% and 45% buffered with Ham's F-10 buffer with 10 mmol/L HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) at pH 7.2. After centrifugation for 15 minutes at 600 g, the sperm pellet was rinsed twice at 300 g in BWW (Irvine Scientific, La Jolla, Calif.) and supplemented with 1% human serum albumin (Sigma, St. Louis). The sperm concentration was adjusted to 25 million sperm per milliliter with BWW -human serum albumin. Two hundred microliter aliquots of this suspension were added to 100 mul of test peritoneal fluid, and the samples were incubated for 16 to 18 hours in a 5% carbon dioxide incubator. Approximately 110 zona-free hamster ova were added directly to each of the assay samples after they were adjusted to 10 million motile sperm per milliliter. Three hours later the oocytes were removed, rinsed thoroughly in BWW, fixed, and stained for scoring of decondensed sperm heads. One hundred oocytes were scored for each assay point.

Computer-assisted motion analysis was performed with a CellSoft system (Cryo Resources, New York) with setup parameters recommended by the manufacturer. Spermatozoa isolated using discontinuous Percoll gradients were suspended in BWW medium to a final concentration of 25 million per milliliter. A 100 mul aliquot of peritoneal fluid was then added to 200 mul sperm suspension and incubated at 37° C for 60 to 90 minutes in a 5% carbon dioxide incubator. Five microliter samples were then assayed for motion analysis with a Makler chamber.

Several parameters are measured during computer-assisted semen analysis. Linearity is a measure of straightness of sperm trajectories as they swim; it is expressed on a scale of 1 to 10, with 10 being a perfectly straight path. Velocity is the swimming speed of the sperm in micrometers per second; it is expressed as both mean velocity and a cumulative percentage of sperm swimming at velocity intervals of 10 mum per second. Lateral head amplitude measures the oscillating lateral displacement of the sperm head from its midline trajectory, and cross-beat frequency shows the number of times the head is noted to cross the calculated curval mean path in 1 second. ^[21]

Data were analyzed with StatView SEGraphics statistical software (Abacus Concepts, Berkeley, Calif.). Parametric means were compared by the Student t test, whereas the Kolmogorov-Smirnov goodness-of-fit analysis was used for comparing cumulative percentile data. Significance was defined as p < 0.05.

Results

Study and control populations were comparable relative to mean gravidity, parity, and cycle day at laparoscopy.

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Figure 1. Results of zona-free hamster egg penetration test comparing sperm mixed with peritoneal fluid obtained at laparoscopy from women with and without endometriosis (Student t test, p < 0.01, n = 20).

The endometriosis group, however, was noted to be significantly older than the controls (Table I) . Regression analysis, with age as the independent variable, failed to demonstrate significance in any of the measured parameters. Indications for the laparoscopic procedures were comparable between the two groups (endometriosis group: infertility, n = 4, pelvic pain, n = 4, and tubal cautery, n = 2; controls: infertility, n = 2, pelvic pain, n = 4, tubal cautery, n = 4).

A significant difference was found when results of the human sperm hamster egg penetration assay were compared. Sperm mixed with fluid from patients with endometriosis penetrated an average of 22.9 ± 5.31 zona-free eggs, as opposed to 44.4 ± 4.96 for the sperm mixed with peritoneal fluid from control patients (Fig. 1) .

By use of computer-assisted semen analysis, sperm mixed with peritoneal fluid from patients with endome-triosis were noted to have a slower mean swimming velocity, 54.0 ± 1.77 versus 59.2 ± 1.05, p = 0.02, n = 20 (Fig. 2) . An increase in the cumulative percentage of sperm swimming at slower velocities was also found (Fig. 3) . Differences were significant in the <20, <30, and <40 mum per second intervals. There were no significant differences at the higher velocity intervals, in linearity, in lateral head amplitude, or in cross-beat frequency.

A simple correlation analysis was performed with mean sperm swimming velocities and the results of the hamster egg penetration assay. Although there was a trend toward a positive relationship, statistical significance was not achieved (Fig. 4) .

Figure 2. Comparison of mean swimming velocity of sperm mixed with peritoneal fluid from patients with and without endome-triosis (Student t test, p = 0.02, n = 20).

Comment

The pathophysiologic mechanisms behind infertility in patients with minimal endometriosis remains unknown. Sueldo et al., ^[16] after demonstrating a significant impact of peritoneal fluid from patients with endometriosis on hamster egg penetration in a murine sperm model, suggested that the fluid from these patients contains a fertility-inhibiting factor or factors. Data from many of the studies published thus far suggest that this factor may be located in the cell-free fraction of the peritoneal fluid and is probably heat labile. Most investigations using the cellular fraction or heat-treated peritoneal fluid were unable to demonstrate an impact on fertility. This study provides more evidence for this fertility-inhibiting factor and its ability to adversely affect the fertilization process. A 32 kd protein, which would presumably have these characteristics, was recently shown to predominate in peritoneal fluids from women with endometriosis. ^[22]

This study contributes additional information to our understanding of the effects of minimal endometriosis on the reproductive process. On the basis of the work of previous investigators, we were able to control for several confounding variables noted in the past. Participants were selected solely on the basis of the presence or absence of stage I disease, and both groups included several patients with proved fertility. Participation was also limited to those women with an otherwise normal pelvis. The cell-free fraction of the fluid was used, no heat inactivation was done, and the sperm samples were washed to remove the seminal plasma, thus more closely simulating the sperm environment in the upper female genital tract. The samples were all analyzed on the same day, under

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Figure 3. Swimming velocity by computer-assisted semen analysis for sperm mixed with peritoneal fluid obtained at laparoscopy from women with and without endometriosis. Asterisk, p < 0.05, Kolmogorov-Smirnov goodness of fit, n = 20.

Figure 4. Plot of correlation analysis between mean swimming velocity by computer-assisted semen analysis and number of eggs penetrated with hamster egg penetration assay (correlation coefficient 0.4392, p = 0.053, n = 20).

identical conditions, and with the same donor sperm sample.

The study and control groups were comparable except with respect to age. Because the incidence of endome-triosis increases with increasing age, it stands to reason that the endometriosis group would, on average, be older. It is well known, however, that fecundity decreases with increasing age and it is possible that this represents a

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confounding variable. Future investigations should probably use age-matched controls.

The samples mixed with fluid from patients with endometriosis had both a significant decrease in mean swimming speed and a significant increase in the proportion of slow swimming sperm. These changes have been shown to correlate well with poor fertilization performance. ^{[23] [24] [25]} The importance of linearity, lateral head amplitude, and cross-beat frequency has not yet been established. The trend toward a positive correlation between hamster egg penetration and sperm velocities may indicate that the same substance or substances are having an impact on both aspects of the fertilization process. Further investigation is required in this area.

In conclusion, we have demonstrated that peritoneal fluid from women with minimal endometriosis has a negative impact on both sperm function and sperm kinematics in an in vitro model.

REFERENCES

1. Drake TS, Metz SA, Grunert GM, O'Brien WF. Peritoneal fluid volume in endometriosis. Fertil Steril 1980;34:280-1.

2. Chacho KJ, Stronkowski Chacho M, Andresen PJ, Scom-megna 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.

3. Drake TS, O'Brien WF, Ramwell PW, Metz SA. Peritoneal fluid, thromboxane B₂ and 6-keto-prostaglandin F_1alpha in endometriosis. Am J Obstet Gynecol 1980;140:401-4.

4. Fakih H, Baggett B, Holtz G, Tsang KY, Lee JC, Williamson HO. Interleukin-1: a possible role in the infertility associated with endometriosis. Fertil Steril 1987;47:213-7.

5. Eisermann J, Gast MJ, Pineda J, Odem RR, Collins JL. Tumor necrosis factor in peritoneal fluid of women undergoing laparoscopic surgery. Fertil Steril 1988a;50:573-9.

6. 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.

7. Fazleabas AT, Khan-Dawood FS, Dawood MY. Protein, progesterone and protease inhibitors in uterine and peritoneal fluids of women with endometriosis. Fertil Steril 1987;47:218-24.

8. Hemmings R, Miron P, Falcone T, Bourque J, Lepage N, Langlais J. Platelet-activating factor acetylhydrolase activity in peritoneal fluids of women with endometriosis. Obstet Gynecol 1993;81:276-9.

9. Halme J, Becker S, Hammond MG, Raj MHG, Raj S. Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am J Obstet Gynecol 1983;145:333-7.

10. Muscato JJ, Haney AF, Weinberg JB. Sperm phagocytosis by human peritoneal macrophages: a possible cause of infertility in endometriosis. Am J Obstet Gynecol 1982;144:503-10.

11. Stone SC, Himsl K. Peritoneal recovery of motile and nonmotile sperm in the presence of endometriosis. Fertil Steril 1986;46:338-9.

12. Curtis P, Lindsay P, Jackson AE, Shaw RW. Adverse effects on sperm movement characteristics in women with minimal and mild endometriosis. Br J Obstet Gynaecol 1993;100:165-9.

13. Burke RK. Effect of peritoneal washings from women with endometriosis on sperm velocity. J Reprod Med 1987;10:743-6.

14. Bielfeld P, Graf MA, Jeyendram RS, De Leon FD, Zaneveld LJD. Effects of peritoneal fluids from patients with endometriosis on capacitated spermatozoa. Fertil Steril 1993;60:893-6.

15. Halme J, Hall JL. Effect of pelvic fluid from endometriosis patients on human sperm penetration on zona-free hamster ova. Fertil Steril 1982;37:573-6.

16. 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.

17. Hinting A, Comhaire F, Schoonjans F. Capacity of objectively assessed sperm motility characteristics in differentiating between semen of fertile and subfertile men. Fertil Steril 1988;50:635-9.

18. Hall JL. Relationship between semen quality and human sperm penetration of zona-free hamster ova. Fertil Steril 1981;35:457-63.

19. American Fertility Society. Revised classification of endometriosis. Fertil Steril 1985;43:351-2.

20. Rogers BJ, Van Campen H, Ueno M, Lambert H, Bron-son R, Hale R. Analysis of human spermatozoa ferti-lizing ability using zona-free ova. Fertil Steril 1979;32:664-70.

21. Vantman D, Koukoulis G, Dennison L, Zinaman M, Sherins RJ. Computer-assisted semen analysis: evaluation of method and assessment of the influence of sperm concentration on linear velocity determination. Fertil Steril 1988;49:510-5.

22. Northnick WB, Curry TE, Muse KN, London SN, Vernon MW. Detection of a unique 32-kd protein in the peritoneal fluid of women with endometriosis. Fertil Steril 1994;61:288-93.

23. Holt WV, Moore HDM, Hillier SG. Computer-assisted measurement of sperm swimming speed in human semen: correlation of results with in vitro fertilization assays. Fertil Steril 1988;44:112-9.

24. Aitken RJ, Best FSM, Richardson DW, Djahandakhch O, Mortimer D, Templeton AA, et al. An analysis of sperm function in cases of unexplained infertility: conventional criteria, movement characteristics, and fertilization capacity. Fertil Steril 1982;38:212-21.

25. Marshburn PB, McOntire P, Carr BR, Byrd W. Spermatozoal characteristics from fresh and frozen donor semen and their correlation with fertility outcome after intrauterine insemination. Fertil Steril 1992;58:179-86.

Editors' note:

This manuscript was revised after these discussions were presented.

Discussion

D r.aul P K aplan,Portland, Oregon. Dr. Nakayama and his colleagues have presented a well-designed controlled study evaluating laparoscopic peritoneal fluid samples from patients with minimal pelvic endometriosis (American Fertility Society stage I) and their effects on donor sperm function in vitro. By use of both the sperm-penetration assay or hamster egg test and computer-assisted semen analysis, this study adds substantially to the growing evidence that peritoneal fluid from patients with endometriosis demonstrates negative effects on sperm function compared with controls.

The sperm-penetration assay or hamster egg test remains a valuable yet controversial assessment of human sperm function in vitro. Since the demonstration in 1976 by Rogers ^[1] that zona-free hamster eggs could be successfully penetrated by human sperm, the hamster egg has become the best available mammalian surrogate for evaluating fertilizing potential in suspected male factor

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infertility. Shy et al. ^[2] in 1988 demonstrated a 68% pregnancy rate after normal sperm-penetration assay results compared with a 27% pregnancy rate after abnormal sperm-penetration assay results in couples with male factor infertility followed up for 3 years. However, there is still no widely accepted standardization of the sperm-penetration assay in all laboratories, and test results are known to vary according to a large number of factors, ranging from abstinence time before sperm collection to composition of the assay medium. ^[1] Rogers ^[1] defined a normal (fertile) result as 10% of eggs penetrated. By these criteria, both groups in Dr. Nakayama's presentation would fall within the normal fertility ranges.

Computer-assisted semen analysis allows the precise measurement of sperm motility. Sperm velocity analysis by this method has previously been shown to predict in vitro fertilization results with 75% accuracy. ^[3] This study demonstrates a small but significant reduction in sperm swimming speed in the endometriosis group but shows no difference in other criteria of sperm motion, including lateral head amplitude, linearity, or cross-beat frequency.

As outlined in Dr. Nakayama's presentation, several previous studies have suggested a cell-free, heat-labile factor to explain the impairment in sperm-oocyte interaction found in the peritoneal fluid of women with endometriosis. ^{[4] [5] [6]} Nothnick et al. ^[7] in Kentucky have recently identified a 32 kd protein called EPF-32 in the peritoneal fluid of 18 of 19 patients with endometriosis but in only 2 of 13 controls. Although as yet unproved, this finding could be a first step in the understanding of the negative effect of peritoneal fluid from women with endometriosis and may eventually result in a much needed marker for this disease.

Dr. Nakayama and his group are to be congratulated for a study with many strengths. The design is well controlled for gravidity, parity, and cycle day of fluid collection. The sperm-penetration assay results show a strong effect ( p < 0.01) in spite of a relatively small number of patients. Because only 4 of the 10 study group patients were infertile and another 2 were having tubal ligations, it is inviting to speculate that the effects would have been even greater if all 10 patients with endometriosis were undergoing evaluation for primary infertility. As stated by Dr. Nakayama, the difference in ages between the two groups is problematic, and age-matched controls would have strengthened their analysis.

The role of minimal or mild endometriosis as a causative agent in infertility remains one of the major con-troversies in our field. A significant improvement inpregnancy rates after medical or surgical treatment ofearly-stage endometriosis has not been conclusively demonstrated in spite of numerous studies in this area. ^{[8] [9]} The majority of cases of minimal endometriosis in an infertility population are unsuspected and are discovered at a diagnostic laparoscopy in the course of a routine infertility workup. Since the addition of the carbon dioxide laser to laparoscopy, it has been our practice to remove all visible endometriotic implants at the time of this initial laparoscopic procedure. Dr. Nakayama and his associates add further significant data to support the theoretic benefits of this type of treatment approach.

In light of this discussion, I would like to ask the following questions. (1) Donor sperm samples will vary considerably in sperm-penetration assay results. Did your group make any attempt to correct for variation between donors or between samples from the same donor in this study? (2) Given your conclusions in this study, what would be your recommendations for the treatment of infertility patients with minimal endometriosis? Are you aware of any clinical data that support recommendations? (3) Although I realize the numbers would be very small, were you able to see any trend in results in the infertile study patients with endometriosis as opposed to those with pelvic pain or tubal sterilization?

REFERENCES

1. Rogers BJ. The sperm penetration assay: its usefulness reevaluated. Fertil Steril 1985;43:821-38.

2. Shy KK, Stenchever MA, Muller CH. Sperm penetration assay and subsequent pregnancy: a prospective study of 74 infertile men. Obstet Gynecol 1988;71:685-90.

3. Holt WV, Moore HD, Hillier SG. Computer-assisted measurement of sperm swimming speed in human semen: correlation of results with in vitro fertilization assays. Fertil Steril 1985;44:112-9.

4. Chacho KJ, Stronkowski Chaho M, Andresen PJ, Scommegna A. Peritoneal fluid in patients with and without endome-triosis: prostenoids and macrophages and their effect in the spermatozoa penetration assay. Am J Obstet Gynecol 1996;154:1290-9.

5. Halme J, Becker S, Hammond MG, Raj MHG, Raj S. Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am J Obstet Gynecol 1983;145:333-7.

6. 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.

7. Nothnick WB, Curry TE, Muse KN, London SN, Vernon MW. Detection of a unique 32-kd protein in the peritoneal fluid of women with endometriosis. Fertil Steril 1994;61:288-93.

8. Beyer SR, Seibel MM, Saffan DS, et al. The efficacy of danazol treatment for minimal endometriosis in an infertile population: a prospective randomized study. J Reprod Med 1988;33:179-83.

9. Olive DL, Marton DC. Treatment of endometriosis-associated infertility with CO₂ laser laparoscopy: the use of one-and-two-parameter exponential models. Fertil Steril 1987;48:18-23.

Discussion (continued)

D r.D avid A damson,Palo Alto, California. Although no studies have shown an improvement in pregnancy rates with medical or surgical treatment of minimal or mild endometriosis, it is important to remember these patients have reduced fertility. Could Dr. Nakayama comment on the level of reduced fertility in his patients with minimal or mild endometriosis? It may be that not all of the endocrinologic or immunologic problems with endome-triosis are in the pelvis. Recent work suggests that reduction of endometrial beta₃ integrins may be associated with reduced implantation rates in patients with endometriosis. Does Dr. Nakayama have any studies underway evaluating endometrial abnormalities associated with endometriosis?

D r. J ames M errill,Moraga, California. Have you studied the peritoneal fluid from any patients with other forms of pelvic pathologic disorders, such as advanced endometriosis, inflammatory disease, or patients with unexplained infertility?

D r. K enneth B urry,Portland, Oregon. I would like to

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bring up the integrin issue. It is known that integrins, which are dimer proteins and therefore heat labile, are required for sperm head binding to the egg membrane. Additionally, there is another group of proteins called disintegrins, which compete for this binding. Do you think that a disintegrin may be involved in endometriosis, and are you looking for such a compound?

D r. B ill Y ee,Long Beach, California. Dr. Nakayama speculated that the peritoneal fluid is effecting sperm motility and egg penetration. From the annual report of the Society of Assisted Reproductive Technology we know that the success rate with gamete intrafallopian transfer is equally good in endometriosis, mild peritoneal adhesions, or unexplained infertility. Could you speculate why this might be from the data presented?

D r. N akayama(Closing). With regard to the variation of sperm donors, the heart of our project was to try to eliminate all confounding variables. However, we were not quite successful with age. Prior data concerning the effect of endometriosis on sperm motility and function has been conflicting. All samples were batched and run together under the same conditions with the same sperm donor. Just to be safe, instead of using 40 to 50 oocytes per assay point, we used 100. That multiplies out to 2000 oocytes at one time, which translates to approximately 40 hamsters killed at one time. I don't think that we would ever want to do that again.

Several people have noted that minimal and mild endometriosis really do not need therapy. Articles dating back to 1981, with the latest from 1992, ^[1] have shown that there is no difference in conception rates between expectant, medical, or surgical management. We did nothing to explain this phenomenon. We used expectant management. I believe that pregnancies were delayed a bit, but ultimately the pregnancy rate will be the same as with other therapeutic approaches.

The data were not analyzed or compared between infertility patients with endometriosis and those with pelvic pain and tubal sterilization. Our objective was to use endometriosis as the primary criterion, not fertility. There are so many contributing factors to the process of infertility and we wanted to keep the study as clean as possible. However, when we go home, we will sit down and try the calculation to see if there is any signifi-cance.

Dr. Adamson and Dr. Burry both talked about integrin as a possible fertility-inhibiting factor. My understanding is that this substance is found in almost every cell in the body and has to do with cell-to-cell adhesion. Investigators have found integrin to be associated with endometriosis implants. We are not doing any research with integrin, but we are going to look at it now that we feel fairly comfortable with our degree of control.

Dr. Merrill, we did not look at pelvic inflammatory disease or advanced endometriosis. We were trying to focus our project on minimal and mild endometriosis.

Dr. Yee, sperm motility and function may have a common inhibiting factor that may have an effect on gamete intrafallopian transfer. If there is a peritoneal factor, then why should gamete intrafallopian transfer work? The egg is harvested straight from the ovary and incubated with sperm. The egg is not bathed in the peritoneal fluid for any considerable period of time before it gets to the sperm. It is then mechanically deposited into the tube. In vivo 5 to 10 sperm would surround the ovum. With this procedure an artificial situation exists in which 200,000 to 500,000 motile sperm surround the egg. It works because of peritoneal fluid dilution and the overwhelming number of sperm.

REFERENCE

1. Inoue M, Kobayashi Y, Honda I, Awaji H, Fujii A. The impact of endometriosis on the reproductive outcome of infertile patients. Am J Obstet Gynecol 1992;167:278-82.

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