Characterization of ciliary activity in distal Fallopian tube biopsies of women with obstructive tubal infertility

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1 Human Reproduction vol.13 no.11 pp , 1998 Characterization of ciliary activity in distal Fallopian tube biopsies of women with obstructive tubal infertility Z.Leng 2, D.E.Moore 1,5, B.A.Mueller 3,4, C.W.Critchlow 3,4, D.L.Patton 1, S.A.Halbert 1 and S-P.Wang 2 1 Reproductive Endocrinology, Department of Obstetrics and Gynecology, Suite 305, 4225 Roosevelt Way NE, University of Washington, Seattle, WA 98105, 2 Department of Pathobiology, 3 Department of Epidemiology, School of Public Health and Community Medicine, University of Washington, Seattle, WA and 4 Public Health Science Division, Fred Hutchison Cancer Research Center, Seattle, WA 98104, USA 5 To whom correspondence should be addressed Biopsy specimens were obtained from the distal end of the Fallopian tubes of 62 women with tubal infertility and examined by light and electron microscopy. Ciliary beat frequency (CBF) measurements were obtained using laser light spectroscopy. Neither demographic nor behavioural characteristics nor serological evidence of past chlamydial infection were associated with CBF measurements. In contrast, CBF were significantly lower (P < 0.05) in tissues with oedema compared to tissues without oedema (6.7 versus 12.9) and in tissues with erythema compared to tissues without erythema (9.2 versus 13.7). Furthermore, CBF measurements did vary by chlamydial serotype pattern, with lower values observed among the tissues of women with antibodies to serotype C or E (without D) as compared to the tissues of women with other serotypes (P < 0.04). However, these data must be interpreted with caution as the numbers of subjects with chlamydial antibodies to serotype C (n 3) or E without D (n 5) were few in number and serotyping of IgG antibodies in blood is not as accurate as it is in bacterial isolates. Confirmation of the suggested association between chlamydial serotype and risk of adverse sequelae could indicate potential new avenues for vaccine research. Key words: chlamydia/chlamydia trachomatis/fallopian tube cilia/infertility/tubal infertility Introduction Salpingitis is followed by an increased risk of distal obstruction of the Fallopian tubes, ectopic pregnancy and pelvic pain among women of reproductive age. The natural history of salpingitis includes not only obstruction of the Fallopian tube at its distal end, but also deciliation (Patton et al., 1983, 1987; Weström et al., 1993). Typically after the tubes have been damaged by salpingitis, the cilia are markedly reduced both in number and quality, with the loss appearing to be permanent (Donnez et al., 1984). The laser light scattering spectroscope was developed to measure ciliary beat frequency (CBF) in Fallopian tubes (Lee and Verdugo, 1976). Recently, a similar spectroscope has been used in vivo in the operating room, to assist the surgeon in determining the degree of damage and thereby the prognosis of reparative surgery (Paltieli et al., 1995). A study of 50 women with distally obstructed Fallopian tubes suggests the identification of mucosal adhesions and the size of the hydrosalpinx are alternative prognosticators (Vasquez et al., 1995). Such information might help infertile couples decide between reparative surgery or in-vitro fertilization, depending on the degree of damage. The purpose of this study was to identify factors that might help to predict the prognosis of the infertile woman with distally obstructed Fallopian tubes with permanently and severely damaged mucosa. Because distal obstruction of the Fallopian tubes is almost always due to an infectious aetiology, we compared mean CBF between women with and without a selfreported history of pelvic inflammatory disease (PID), and for those with and without serological evidence of past Chlamydia trachomatis infection. We also assessed the relationship between CBF and histopathological characteristics, contraceptive use histories, and personal and demographic characteristics identified in a detailed personal interview. Materials and methods Subjects Since we were trying to identify factors important to the prognosis of the woman with tubal infertility, our comparison group also consisted of infertile women with distally obstructed Fallopian tubes but who lacked the factor being studied. The group of women in this study were part of a larger study of silent pelvic inflammatory disease (PID), and were aged years, had radiographic or laparoscopically verified distally obstructed Fallopian tubes, and sought infertility care from physicians in the greater Seattle area during the years They were identified prior to surgical repair of their Fallopian tubes. Women were informed of the study by mail, and then were contacted by telephone 10 days later. If they agreed to participate in the study, an in-person structured interview to obtain data concerning personal characteristics and past exposures was conducted. At the time of surgery, which was generally performed a few days later, a 3 10 mm biopsy was obtained from the distal end of one or both Fallopian tubes, but only if an incision was made in the distal end of the tube as part of the surgical repair. The research coordinator attended each surgery to record observations and measurements and to obtain a blood sample. Medical records of consenting women were abstracted for details of their infertility investigation. Both the research coordinator and the chart reviewer were blinded to information obtained in the interview. A total of 167 European Society of Human Reproduction and Embryology 3121

2 Z.Leng et al. women thought to have at least one distally obstructed tube and who were planning surgical repair agreed to an interview; of those, distal tubal tissue was obtained from 91. Women with concurrent moderate to severe endometriosis were excluded from the study because of the possibility that their tubal obstruction arose via a non-infectious pathway. Since phimosis is thought to be on a continuum to distally occluded Fallopian tubes, women with phimosis were included in the study. Distal tubal biopsies were obtained for laser light spectroscopy from 62 women with distal obstruction or phimosis and are the focus of the present analysis. This protocol and the consent signed by each subject was approved by the University of Washington Human Subjects Review Committee and by the other area hospitals involved. Tissue handling Tubal tissue biopsies obtained at surgery were divided into four sections for scanning and transmission electron microscopy, light microscopy and laser spectroscopy. Mucosal surfaces of the fresh tissues were evaluated under magnification of a dissecting microscope for proper orientation of the tissues prior to preparation for electron microscopy or CBF assays. This ensured that epithelium was observed when the CBF measurements were taken. The tissues for laser spectroscopy were placed in Rose chambers and bathed in Eagle s medium using tissue culture procedures (Patton et al., 1989). CBF measurements were taken on the day of preparation or after an overnight incubation at 37 C. During the initial studies of the laser light spectroscopy no discernable differences were seen in CBF measurements taken the day of preparation or after an overnight incubation in our laboratory. All microscopic and spectroscopic measurements were obtained by a single observer (D.L.P. for the former and a spectroscopic technician for the latter) who was blinded to the clinical and interview data. Laser light spectroscopy The laser light scattering system used to measure CBF was described previously (Holloway et al., 1988). Briefly, monochromatic light from a 5 mw He-Ne laser was directed through an optical fibre to illuminate the mucosal surface of the tissue. Light back-scattered from the tissue was conducted through a parallel optical fibre to a photodiode, and spectral analysis of the resultant electronic signal was performed using computer software (ASYST; MacMillan Software). In earlier studies conducted in our laboratory (Patton et al., 1989), CBF was measured using a different laser light scattering system (Lee and Verdugo, 1976), which employed an inverted light microscope rather than fibreoptics to direct light to and from the specimen. Equivalence of the two systems was demonstrated in independent studies (Halbert et al., 1990). Depending on the measurement variance, five to ten readings were recorded per piece of tissue. The average of these readings reported in beats/s or Hz was used for analysis. Only non-zero readings were recorded, unless all areas examined gave zero readings, in which case a value of zero was entered for that piece of tissue. Therefore, the non-zero scores reflect readings taken only from ciliated areas that had some motion. If more than one distal tubal specimen was available from the same study subject, the mean CBF was measured for both, and the reading from the right distal site was used in the data analysis. In this way, a single mean CBF measurement was used for each subject. When analyses were restricted to only right or left distal specimens, the results did not differ. Light microscopy Routine histology was assessed from formalin-fixed paraffin-embedded sections stained with haematoxylin eosin. The presence of 3122 inflammatory cells such as plasma cells, monocytes, and polymorphonuclear neutrophils was noted and considered significant if there were five or more per high power field. The identification of plasma cells was enhanced by staining with methyl green pyronine. A sample was rated positive for oedema if greater than half of the observed area was markedly swollen, with fluid deposition within the connective tissue of the submucosal compartment. Tubal tissue was scored positive for erythema if the submucosa contained engorged capillaries and/or inflammatory infiltrate within the submucosa not associated with capillaries. Electron microscopy Tissues were fixed and prepared for electron microscopy as previously described by Patton et al. (1989). Morphological damage was scored on scanning electron microscopy on a scale from 0 to 9, with 0 representing normal mucosal folds, normal ciliation and normal microvilli on secretory cells. A 9 was assigned if there were no mucosal folds, complete deciliation and no microvilli on the secretory cells and pitting of the surface epithelium. Scores between 0 and 9 represented gradations between these extremes (Lee and Verdugo, 1976). Determination of C.trachomatis antibody titre Each blood sample was first screened by microimmunofluorescence for immunoglobulin IgM and IgG antibody at 1:8 dilution against a selection of pooled C.trachomatis antigens (CJHI, A, BED, GF, and K) (Wang et al., 1984). Each positive serum was further titrated at 2-fold dilutions with individual serovar antigens to obtain an endpoint titre. Patients with a serovar-specific IgM or IgG antibody titre of 1:8 were considered to have a positive C.trachomatis antibody response. Interview questionnaire A detailed, structured questionnaire was used to obtain information from study subjects during an in-person interview, usually in the home. Reproductive and contraceptive histories were obtained, as well as demographic data. The timing and duration of events were elicited for these variables as well as information concerning habits and practices such as cigarette smoking and douching. The women were asked if they had ever had an infection or positive culture for C.trachomatis, gonorrhoea or other sexually transmitted diseases. A self-reported history of PID was obtained by asking women whether or not a physician had ever told them they had a deep pelvic infection unrelated to the bladder or vagina. For all self-reported data, care was taken to obtain only information about events occurring prior to each woman s reference date, that is the date when she first began attempting conception without success. In this manner we sought to avoid biasing our results by the inclusion of findings based on diagnostic tests and procedures conducted as part of her infertility work-up. Statistical analysis All historic events identified by the interview were dated relative to the time the woman first started trying to conceive (reference date) except for age, which was at the time of surgery. Mean CBF were computed for the entire population, followed by stratification on the characteristics of interest. Standard statistical tests were performed to test differences between subgroups using Student s t-test or the Mann Whitney U-test for continuous data and χ 2 -analysis or Fisher s exact test for categorical data. Results The average age at the time of surgery of women included in the study population was (mean SEM) years,

3 Ciliary activity in distal Fallopian tube biopsies Table I. Mean ciliary beat frequency (CBF) of 62 women with distally obstructive tubal infertility by selected demographic, behavioural, and contraceptive characteristics reported in the personal interview Characteristic n (%) CBF (beats/s) P value a Mean SE All women Age (years) (21.0) (40.3) (22.6) (16.1) Smoking status b Non-smokers 24 (40.0) Smokers 36 (60.0) No. of prior sex partners c 1 9 (14.8) (19.7) (29.5) (16.4) (19.7) Self-reported history of PID c No 38 (62.3) Yes 23 (37.7) Oral contraception c Never used 15 (24.6) Ever used 46 (75.4) IUD history c Never used 41 (66.1) Ever used 21 (33.9) Type of IUD used Dalkon Shield 9 (14.5) Other IUD d 12 (19.4) a Based upon χ 2 -analysis. b Missing data for two subjects. c Pelvic inflammatory disease; missing data for one subject. d Includes one subject who had also used a copper intrauterine device (IUD), with CBF No other subject in this study had ever used a copper IUD. with 84% of the women 38 years of age (Table I). Sixtytwo per cent of women reported no history of PID prior to the reference date, 60% were cigarette smokers, and 36% had 10 lifetime sexual partners. The CBF of distal Fallopian tube tissues obtained from all subjects ranged from 0.0 to 21.7 beats/s, with a mean CBF of beats/s (mean SEM). CBF readings did not vary by age, cigarette smoking, number of prior sexual partners, history of PID, or prior use of oral contraceptives and intrauterine contraceptive devices (IUD) (Table I). In addition, no association was observed between CBF measurements and type of IUD (Table I), douching, and use of barrier contraceptive methods (data not shown). Ten of 57 women (17%) reportedly had been told by a physician that they had an infection with C.trachomatis (Table II). However, 41 (68%) of 60 women had IgG antibodies to C.trachomatis, suggesting previous C.trachomatis infection. CBF measurements were similar for tissues from women with and without a self-reported history of chlamydial infection ( versus ), and with and without positive IgG chlamydial serology ( versus , P 0.18). Furthermore, there was no apparent relationship between CBF measurements and increasing serum levels of IgG antibody (data not shown). The most common IgG serotype pattern, BED (B-group), Table II. Mean ciliary beat frequency (CBF) of 62 women with distally obstructive tubal infertility by self-reported history and by serological evidence of Chlamydia trachomatis infection C.trachomatis n (%) CBF (beats/s) P Mean SE value a Self-reported history of C.trachomatis b No 47 (82.5) Yes 10 (17.5) C.trachomatis serology IgG antibody c Negative ( 1:8) 19 (31.7) Positive ( 1:8) 41(68.3) IgG serotype d J 6 (14.6) H 2 (4.9) I 1 (2.4) 15.7 CHI, CJHI 3 (7.3) C-group 12 (29.3) B 1 (2.4) 11.3 BED, ED, D 14 (34.2) BE, E 5 (12.2) B-group 20 (48.8) F 3 (7.3) GF-group 3 (7.3) Multiple (Mul) 6 (14.6) a Based upon χ 2 -analysis. b Missing data for five subjects. c Missing data for two subjects. d Performed for the 41 women with detectable C.trachomatis antibody titres. was observed in 20 (49%) of the 41 women with antibodies to C.trachomatis, followed by CJHI (C-group) in 12 (29%), and F (GF-group) in three (7%). There were six (15%) women who had IgG antibody broadly reactive to multiple serovar antigens (see Table II). The lowest CBF (0.0 beats/s) was measured in tissues from the three women who had serotype patterns containing C (either CHI or CJHI). This was significantly lower than that observed for other serotypes combined ( , P 0.02) and lower than that in the 19 women without IgG antibodies to C.trachomatis ( , P 0.01). The second lowest mean CBF ( beats/s) was measured in tissues from the five women who had serotype patterns containing E (but not D). This CBF mean value was significantly lower than that observed for other sertoypes combined ( , P 0.037, n 36) and lower than that in women without IgG antibodies to C.trachomatis (P 0.009). CBF measurements in the tissues of women with serotypes other than C or E (without D) as well as without IgG antibodies to C.trachomatis were similar. CBF was lower in tissues with erythema (P 0.03) or oedema (P 0.02) than in those without such damage (Table III). The presence or absence of plasma cells (five or more per high power field) as read by light microscopy (data not shown) and cilia scores based on scanning electron microscopy were not associated with CBF measurements (Table III). Interestingly, 22% of women had oedema, 57% had erythema, 26% had a significant number of plasma cells (five or more per high power field), and 70% had moderately to severely damaged cilia (score on scanning electron microscopy of 2.0 or higher). 3123

4 Z.Leng et al. Table III. Mean ciliary beat frequency (CBF) of 62 women with distally obstructive tubal infertility by histopathology findings Characteristic n (%) CBF (beats/s) P value a Mean SE Oedema b No 42 (77.8) Yes 12 (22.2) Erythema b No 23 (42.6) Yes 31 (57.4) Ciliated cell scores c 0 1 (2.3) (2.3) (13.6) (11.4) (29.6) (18.2) (22.7) a Based upon χ 2 -analysis. b Insufficient tissue was available in eight subjects for light microscopy. c Insufficient tissue was available in 18 subjects for scanning electron microscopy. Discussion Ciliated epithelial cells are an integral part of the mucosa of the Fallopian tubes. At the fimbriated end of the tube ~60% of the cells are ciliated (Donnez et al., 1984). Approximately cilia are at the apex of each ciliated cell. Approximately 12 14% of these ciliated cells lose their cilia during the luteal phase of the menstrual cycle, presumably due to progesterone, and then regenerate them during the early follicular phase, presumably in response to oestrogen (Verhage et al., 1979). However, as a result of inflammation or salpingitis, deciliation, which is thought to be permanent (Donnez et al., 1984), increases with increasing severity of the tubal damage and can be quite extensive. Since cilia are important for normal ovum transport (Odor et al., 1973), such permanent loss of cilia from individual cells may contribute to infertility, tubal pregnancy and influence the degree of success after surgical repair (Vasquez et al., 1983). Recently Paltieli et al. (1995) studied ciliary activity in human Fallopian tubes using an invivo laser scattering instrument and found a high positive correlation between CBF and the percentage of ciliary cells in the fimbria and in the ampulla by scanning electron microscopy. We did not find such a correlation in our study. This discrepancy may be due to several differences between the two studies. In our study we did not record CBF readings from areas on the tissue where the signal was not above background; by ignoring such silent areas and recording only positive readings, higher readings overall would be expected. Secondly, 28 of the 31 subjects in the study by Paltieli et al. presumably had normal Fallopian tubes and only three of their subjects had mechanical infertility in contrast to all of our subjects. Furthermore, the site of the obstruction, if any, in the study by Paltieli et al. was not defined whereas all of our subjects had distally occluded Fallopian tubes. This study demonstrates decreased ciliation and low CBF in the surviving cilia in the mucosa of distally obstructed Fallopian tubes, similar to previous findings from our laboratory (Patton et al., 1989). Our previous study also found decreased 3124 numbers of cilia and a decrease in CBF in the Fallopian tubes of women with distally obstructive tubal infertility compared with women with normal Fallopian tubes. In the current study, we include data obtained via an in-depth interview, in addition to comparing women with and without previous chlamydial infection among those with distally obstructed Fallopian tubes. Our results are consistent with previous studies reporting anatomic reduction in cilia in humans and pig-tailed macaques with salpingitis or its sequela, tubal infertility, as measured by scanning electron and light microscopy (Donnez et al., 1984; Patton et al., 1983, 1987). Although we observed decreased ciliary activity as measured by mean CBF with increasing age, this trend was not significant. We also did not observe significant variation in mean CBF by other personal characteristics, including smoking, contraceptive use and numbers of sexual partners. These characteristics have previously been shown to be associated with tubal damage in studies comparing women with and without tubal damage (Daling et al., 1985; Mueller et al., 1992; Scholes et al., 1993). However, it is possible that in our study, which evaluated only women with distally obstructive tubal disease, the tubes were so severely damaged that differences in CBF by these demographic or behavioural characteristics could not be distinguished. The prevalence of C.trachomatis serotypes varies by geographical region. The serotypes we observed in this study (B-group 49%, C-group 29%, F-group 7% and multiple 15%) are consistent with those reported among women with acute pelvic infection attending a sexually transmitted disease clinic in Seattle (Kuo et al., 1983) and in Rotterdam (van Duynhoven et al., 1998). In the current study evaluating late sequelae of salpingitis, we observed a potentially different pathological impact on the distal Fallopian tube associated with certain serotypes. Any combination of serotypes containing serotype C demonstrated no ciliary activity. In fact, in all subjects with tubal tissues obtained during the study period , any serotype pattern containing C was always associated with a zero CBF reading (n 5, P compared with the CBF readings with other serotypes; the additional two subjects were one with phimosis and one with proximal obstruction whose tissues were only obtained from the proximal part of the tube and therefore were not included in this study). Although the numbers are small and need verification in future studies, these findings suggest that some serotypes may cause more severe long-term degenerative changes of the Fallopian tube resulting in a poorer prognosis after surgical repair. Serotype E was also associated with a lower ciliary activity, but only when not associated with serotype D. This situation is analogous to that of another bacterium, E.coli, O157H7 which causes bloody diarrhoea and even death, unlike other E.coli serotypes that colonize the gastrointestinal tract. Our data suggesting the presence of a specific virulent serotype is in apparent contrast to a study by Persson et al. (1993) that did not demonstrate such an association with a particular serotype. Their study included 12 women with acute salpingitis; they assayed the serotypes of the bacterial isolates directly rather than indirectly through the antibodies, as our study did. Our population, consisting of infertile women with obstructed tubes, was quite different in that it also included women with

5 Ciliary activity in distal Fallopian tube biopsies Figure 1. Scanning electron micrograph from the Fallopian tubes of women with distally obstructed Fallopian tubes at the time of surgery. (A and B) Women with primary infertility, no history of pelvic infection, antibodies to C.trachomatis [1:16, serotype BED for case (A) and 1:64, serotype J for case (B)], no oedema by light microscopy, and inflammatory cells by light microscopy. Deciliated areas and decreased cilia per ciliated cell (c) are demonstrated in (A). In addition, (A) demonstrates extruded ciliated cells (ec). In (B) the secretory cells (s) are almost normal. Ciliary beat frequency was 15.4 beats/s in case A and 20.9 beats/s in case (B). Ciliary score in scanning electron microscopy was 3 in case (A) and 1 in case (B). Original magnification Scale in lower left centre is 1.0 µm. (C) Tissue from a woman (shown for comparison) with normal Fallopian tubes undergoing surgery during the same time period ( ) but not part of the study. Note that there are many more ciliated cells and more cilia per ciliated cell. Original magnification Scale in lower left is 1.0 µm. (D) and (E) are from the same patients as (A) and (B) respectively, and demonstrate by light microscopy areas of monocytes (m) (D) and plasma cells (E). Original magnification 400. prior asymptomatic disease, and perhaps provides a better indication of which serotype is most appropriately targeted for a vaccine to prevent the ultimate sequelae of chlamydial infection. This is particularly relevant when one notes that almost all infertile women with positive serology to C.trachomatis (IgG 1:32) have damaged Fallopian tubes, at least in our laboratory (Moore et al., 1982). Our findings confirm the importance of C.trachomatis in tubal infertility as a sequel to acute salpingitis, similar to that reported in other studies (Mol et al., 1997). Although few women (16%) in our study reported a history of C.trachomatis infection, two-thirds had IgG antibodies to C.trachomatis. This suggests a fairly high prevalence of prior asymptomatic chlamydial infection among women with tubal infertility in 3125

6 Z.Leng et al. our region. This high prevalence emphasizes the importance of routine screening for C.trachomatis, particularly among high risk women, to prevent the sequelae of what is largely a silent process. The observed histopathological changes including deciliation, oedema, erythema, and plasma cells are presumed to be a chronic process, are expected to persist after surgical repair, and may partially explain the low pregnancy rates among these women even after salpingostomy. The presence of plasma cells lends further support to an infectious aetiology of obstructive Fallopian tube infertility. Recently, Minassian et al. (1992) reported an association between severity of acute salpingitis and C.trachomatis titre in women with tubal infertility. However, in our study we did not observe a decrease in mean CBF with increasing titres of C.trachomatis. It is possible that our inability to detect such a trend was due to the small number of women enrolled in our study for whom tissue specimens were available, to differences in methodology, or because we studied only severely diseased tissues. There are limitations of this study. We did not correlate the effect of the menstrual cycle or ovarian steroid levels to the ciliary beat measurements in these subjects; although there was a concerted effort to operate in the follicular phase of the cycle this effect would need to be investigated further before our conclusions can be validated. Because of our small sample size we were unable to further examine associations among relevant patient subgroups, for example women with tubal oedema or erythema to control for other potentially important confounding variables such as number of prior genital infections. History of smoking was characterized relative to the reference date which may have preceded the date of surgery by several years. This may have underestimated the effect of smoking as infertile women are encouraged to discontinue smoking. In addition these results were obtained only from women with obstructive distal tubal infertility who elected surgical repair, and thus cannot be generalized to other populations. Although the IgG chlamydial serology provides evidence of past infection with C.trachomatis, serotyping of the antibody only approximates the specific serotypes of the infecting organisms. It is difficult antigenically to separate closely related serotypes within groups, such as C, J, H and I within the C-group or B, E and D within the B-group, for example, unless the original C.trachomatis organisms have been serotyped. This, of course, would require a prospective study with long-term follow-up. Serological evidence of past infection was limited only to C.trachomatis; serological evaluation of infection with other important organisms that potentially could affect CBF measurements was not performed. The CBF readings of zero on several subjects require further discussion. The tissues prepared for the laser light spectroscope were first viewed by a dissecting microscope for orientation and for the presence of cilia. Areas that gave a zero reading were ignored unless the entire tissue gave no discernable readings. Therefore, an overall reading of zero meant either the whole tissue was denuded of cilia or that the cilia that were present did not register a signal above background. It is interesting that CBF readings from study subjects with any serotype pattern containing C were always zero. In summary, our results represent an evaluation of tubal pathological changes potentially associated with the sequelae of acute salpingitis and thus provide a basis for further research. Acknowledgements The laser light-scattering spectroscopy studies were performed by Richard L.Anderson. The clinical data were collected by Tara Cannava, Sha-Ke Wang, Hanne Thiede and Soe Soe Thwin; the interviews were performed by Jill Ashman and the chart reviews by Dean Coonrod. We acknowledge the following physicians who informed and helped recruit potential subjects for this study: Emmett F.Branigan, Robin E.Cole, Lawrence R.Donohue, Lee R.Hickok, Jack R.Lamey, Michael P.H.Lau, Robert E.Lieppman, Lori A.Marshall, Robert E.McIntosh, Mark C.Rattray, Steven L.Sharmahd, Donald C.Smith, Michael R.Soules, Leon R.Spadoni, Barry C.Stewart, Jane K.Uhlir, and Paul W.Zarutskie. This study was supported in part by National Institute of Health Grants R01 HD and R01 HD References Daling, J.R., Weiss, N.S., Metch, B.J. et al. (1985) Primary tubal infertility in relation to the use of an intrauterine device. N. Engl. J. Med., 312, Daling, J.R., Weiss, N., Spadoni, L.S. et al. (1986) Cigarette smoking and primary tubal infertility. In Rosenberg, M.J. (ed.), Smoking and Reproductive Health. 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