A very sticky riddle

dream. The paper published by Khan et al. in the May 1996 issue of the Journal was an important contribution to fertility research. Doctors Khan, Daya, Collins, and Walter, with their expertise in this area built upon their McMasters' experience, take the straight and narrow--stressing the limitations to crossover studies for pregnancy outcome. Doctor Olive, who is one of our unique experts in the quantitative sciences, points out that "when choosing a trial design one must understand and account for all the nuances of the design, using appropriate an~alytic methods--otherwise no design is adequate." Crossover designs may require fewer subjects, but the analysis of data from a crossover design is affected adversely by patient dropouts, differences at initial baseline, regression to the mean, failure to return to the original baseline before second treatment, and missing data. In that sense the analysis is not as "robust" as compared with a parallel design. In the past the actual design of a crossover trial was obtained by consulting a published table of designs or by following some relatively simple rules. As suggested by Doctor Olive a priori rules and tables may not always be appropriate for the current trial. A more flexible approach is to make the design satisfy the objectives of the trial rather than vice versa. It appears that many of these difficulties and greater degrees of flexibility can be provided by using a computer search algorithm that takes into account the particular features of the specific crossover trial. A computer algorithm of this type has been developed and described by Jones and Donev (1). This program enables designs to be augmented with additional periods or sequences of treatment. The unique advantage o f the latter is that each stage of the trial can be modified so new model assumptions can be incorporated to include information gained during the trial. For example the carry-over effects of clomiphene citrate therapy may be quite different from hMG. Sometimes an investigator may not appreciate certain carry-over effects (such psychological effects) of a particular medication before the initiation of the investigation. The degree of flexibility provided by the computer algorithm for crossover trials allows one to depart from the "standard model" and incorporate specific assumptions about the form of treatment and carry-over effects. The comments of Doctors Mol, Bossuyt, and Daya seem to provide even more flexible "human-derived models" of computer algorithms. The original article and this correspondence has been a gratifying and enlightening experience for an editor. It is the type of topic that would clearly benefit from electronic publishing on World Wide Web, where an even more interactive journal format could be provided. In fact the "Letters to the Editor" section

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Letters-to-the~editor

of journals is ideal for electronic dissemination. It will surely happen as soon as editors overcome the fear of cannibalizing their print sales and learn how to make money on electronic publication. Perhaps down-loading costs for this real-time between Doctors Daya, Olive, Mol and Ananth will be covered by your favorite corporate sponsor. Paul G. McDonough, M.D., Editor, Letters REFERENCE 1. Jones B, Donev AN. Modelling and design of cross-over trials. Stat Med 1996;15:1435-46. A Very Sticky Riddle

To the Editor (Letter 1 of 2): We have appreciated the opportunity to read the recent paper of van der Linden et al. (1) describing the lack of endometrial cell adhesion to an intact amniotic membrane in vitro. Endometrial cell adhesion to the pelvic peritoneum is probably an iraportant and poorly understood step in the pathogenesis of endometriosis. The authors (1) deserve a lot of credit for studying this phenomenon in vitro. However, some aspects regarding the study method and interpretation of the results were unclear and raised several questions. 1. Why were the endometrial biopsies, which were collected during menses, washed? This washing procedure may have resulted in false-negative results by removing the cell-free fraction of the menstrual endometrium, which may be crucial in the process of adhesion to intact pelvic peritoneum in vivo. 2. Why were the endometrial tissue fragments cultured for only 24 hours? This culture time may have been too short to allow adhesion of endometrial cells to intact amniotic membranes. 3. The proposed rationale for washing the suspended amniotic membranes several times with phosphate-buffered saline was to prevent a "false impression of adhesion." How is this "false impression" defined? It is possible t h a t this washing procedure abruptly removed cells t h a t were in an early stage of adhesion or implantation and t h a t this early s t a g e n e e d s to be acknowledged? How can the authors be sure t h a t proper washing is essential to detect true adhesion? They define adhesion as uninterrupted contact between the amniotic membrane and e n d o m e t r i a l cells or fragments. However, interrupted contact between endometrial fragments and the amniotic membrane may be a consequence of the washing procedure itself and does n o t n e c e s s a r i l y exclude true adhesion. 4. The authors mention t h a t it has never been

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described that endometrial tissue can grow on the peritoneal surface and that disruption of the peritoneum is essential for adhesion between endometrial cells and the peritoneal wall (see Discussion). We recently reported that laparoscopic seeding or placing of unwashed menstrual endometrium on top of an intact pelvic peritoneum in baboons resulted in extensive implantation with the development of peritoneal endometriosis after 2 months (2). Superficial scarification of the pelvic peritoneum to disrupt the peritoneal lining caused adhesions but did not favor the implantation of menstrual endometrium (2). These observations showed that in vivo menstrual endometrium can adhere to and grow on an intact pelvic peritoneum.

Thomas M. D'Hooghe, M.D., Ph.D. Leuven University Fertility Center and Department of Obstetrics and Gynecology University Hospital Gasthuisberg Leuven, Belgium Charanjit S. Bambra, Ph.D. Institute of Primate Research Nairobi, Kenya Joseph A. Hill, M.D.¢ Fearing Research Laboratory Division of Reproductive Immunology Department of Obstetrics Gynecology and Reproductive Biology Brigham and Women's Hospital Harvard Medical School Boston, Massachusetts August 17, 1996 REFERENCES 1. van der Linden PJQ, de Goeij AFPM, Dunselman GAJ, Erkens HWH, Evers JLH. Endometrial cell adhesion in an in vitro model using intact amniotic membranes. Fertil Steril 1996;65:76-80. 2. D'Hooghe TM, B a m b r a CS, Raeymaekers BM, de Jonge I, Lauweryns JM, Koninckx PR. Intrapelvic injection of menstrual endometrium causes endomehiosis in baboons (Papio anubis and Papio c3,nocephalus). Am J Obstet Gynecol 1995; 173:125-34.

Reply of the Authors: D'Hooghe and coworkers express their concern regarding some aspects of our in vitro model of the initial phase of endometrium-peritoneum cell-cell contact (1). Because their questions touch upon the core of modeling in endometriosis research, we appreciate the opportunity to respond to their remarks. Endometrial biopsies were washed to remove

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blood cells and clotting factors in order to study selectively cell-cell interaction between the endometrium and the epithelium. After washing, expression of cell adhesion molecules was still present on endometrial epithelial (integrin a2, a3, a4, and a6 subunits, E- and P-cadherins) and stromal (integrin a5 subunit) cells. We agree that, in theory, also fibrin deposition might glue endometrial fragments to the peritoneum. However, under normal circumstances this process will be prevented by fibrinolytic factors in the peritoneal fluid (2). Cell-cell adhesion is a process of hours rather than days. The aim of our study was to investigate the initial steps in the adhesion process. Therefore, we did not culture the cells for more than 24 hours. Because adhesion did occur to the nonepithelial side of the amniotic membrane, we would have to accept that washing would selectively remove endometrial fragments from the intact epithelial side. This does not seem likely given our present knowledge of the interaction of cell adhesion molecules between a cell and its neighboring cell, and between a cell and the extracellular matrix. We assume that defects in the peritoneal lining (microtraumas) and "superficial scarification" as performed by D'Hooghe and coworkers are different entities (3). However, only careful microscopic examination of cross sections of implanting endometrial tissue will allow this conclusion. In this, our in vitro model differs from the baboon model: D'Hooghe and coworkers studied existing implants. Whether these resulted from implantation on intact peritoneum, from implantation on exposed stroma, or even from induced metaplasia cannot be derived from the findings in their model. In conclusion, the absence ofendometrial adhesion to intact amniotic membranes is not enough to prove that implantation is prevented by intact peritoneum. However, also the presence of endometriosis in baboons, several weeks after seeding of endometrium into their peritoneal cavity, is not enough to prove that implantation does occur on intact peritoneum and neither is demonstration of c_ell adhesion molecule expression by epithelial cells from the peritoneal lining in vitro (Witz CA, Montoya IA, Norris CJ, Schenken RS, abstract). Careful combination of data from different models, however, hopefully will allow for further unravelling the mystery of the first few steps in the development of endometriosis.

Paul J. Q. van der Linden, M.D. Anton F. P. M. de Goeij, Ph.D. Gerard A. J. Dunselman, M. D. Johannes L. H. Evers, M. D. Academisch Ziekenhuis Maastricht Letters-to-the-editor

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