SPERM TRANSPORT FROM THE EXTERNAL CERVICAL OS TO THE FALLOPIAN TUBES IN WOMEN: ATIME AND QUANTITATION STUDY*t

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1 FERTILITY AND STERILITY Copyright 1973 by The Williams & Wilkins Co. Vol. 24, No.9, September 1973 Printed in U.S.A. SPERM TRANSPORT FROM THE EXTERNAL CERVICAL OS TO THE FALLOPIAN TUBES IN WOMEN: ATIME AND QUANTITATION STUDY*t DIANE S. FORDNEY SETI'LAGE, M.D., M.S., MASANOBU MOTOSHIMA, M.D., AND DONALD R. TREDWAY, M.D. Department of Obstetrics and Gynecology, Los Angeles County/University of Southern California Medical Center, Los Angeles, California Studies of sperm transport in the human female are few. It probably does not occur except during the late proliferative and ovulatory portions of the menstrual cycle Animal data on subprimate species imply roles in sperm transport to sperm motility, uterine and oviductal contractility, coital thrusting with oxytocin release, seminal plasma constituents, and, possibly, oviductal ciliary movements. 2, 3 Animal data also suggest great depletion in the numbers of deposited sperm which actually reach the oviduct, that transport times may vary from 5 min. to several hours depending on the species and the author, and, in the rabbit, that sperm accumulates in the ampulla, which is the probable site of fertilization Clinical and in vitro studies in the human support the importance of sperm motility in passage through the cervix. 2, 4-8 However, passage of inert particles from the vagina to the oviduct has been described. 9, 10 Similarly, uterine and cervical contractions producing negative pressure, and "insuck" theory, are suggested by clinical studies, although these are denied by others.2, 11 The possibility also exists that seminal plasma contains a factor or factors that increase cervical mucus solubility 2. 12, 13 or enhance any role the uterus might play in passage of Received May 17, * Supported by The Ford Foundation. t Presented as the Squibb Prize Paper at the Twenty Ninth Annual Meeting of The American Fertility Society, April 5-7, 1973, San Francisco, Calif. 655 sperm through the cervical mucus. 2, 3 Certainly prostaglandin E 2, present in large quantities in seminal plasma, increases uterine and, possibly, oviductal contractility. 2, 3 Whether this is essential, enhancing, or inhibiting to sperm transport through the uterus to the oviducts is unknown, as is the level of the female reproductive tract where an obvious effect might be exerted. Despite the bits of information described, data on the physiologic characteristics of sperm transport in the human female reproductive tract are scant under any conditions. Rubenstein et a1. 13 demonstrated motile sperm in the cervix, uterine cavity, and oviducts within 30 min. after insemination. No correlation was made to ovulatory status of patients, quality of inseminates, or phase of the cycle when the study was done. All patients had uterine or tubal pathology. Brown 14 demonstrated sperm transport in excised pathologic utero-oviductal preparations exposed to ejaculates within 69 min., confirming in vitro capacity for transport. Again, the variables mentioned in Rubenstein's work were uncontrolled. Moghissi, 15 however, was unable to demonstrate sperm transport in a similar study utilizing normal excised organs, suggesting that proposed intrinsic factors may be extremely important. Horne and Thibault, 16 as a prognostic tool for cases of infertility, studied the presence or absence of sperm in peritoneal fluid collected at laparoscopy 24 hr. after coitus. Ahlgren,17 more recently, found lower numbers of sperm in the oviductal

2 656 SETILAGE ET AL. Vol. 24 ampullae of normal women than in those with clinical and laparoscopic evidence of oviductal obstruction at undefined times following coitus. Sperm numbers found in tubal washings or aspirations of women with hydrosalpinx were much higher and may indicate that sperm pass through the tube and into the peritoneal cavity in normal women. 17 Faundes et al. 18 utilized oviductal, endometrial, and peritoneal washings from patients requesting sterilization and found sperm in 80% of patients in some part of these washings 7-37 hr. after coitus. No attempt to study sperm transport systematically in the human or nonhuman primate is reported under normal or altered conditions which might clarify any potential mechanisms. The present study concerns itself with characterizing sperm transport under optimal, controlled, assessable conditions. METHODS Subjects selected for this study were women who specifically requested surgical sterilization and volunteered for the project. All subjects were regularly menstruating women who were proven fertile, with no history and/or evidence of reproductive organ abnormalities. Additionally, no subject received hormonal medications or utilized an intrauterine device for a minimum of 1 menstrual cycle preceding her participation in the study. Any otherwise eligible patient for whom the study requirements constituted suboptimal management for social or medical reasons peculiar to her, or who expressed preference for another surgical approach, was excluded from the sample. As the survival time for sperm in the human fallopian tube is not exactly known, all subjects were restricted from coitus for days preoperatively. Daily examinations of cervical mucus and vaginal secretions during this time confirmed the absence of sperm. Previous reports confirm the detection of sperm in cervical mucus up to 96 hr. after coitus and in vaginal secretions for up to 36 hr. 2. 5, 10 Since practical evaluation of sperm transport must be accomplished during the time when conception is likely, the mid cycle status of subjects was determined by the clinical elastoviscosity features of cervical mucus (ferning and spinnbarkheit) and by serum estradiol determinations begun on Day 10 of the operative menstrual cycle. The study was done within 36 hr. of the estradiol preovulatory peak for each subject. Surgical excision of both fallopian tubes was accomplished at predetermined times following insemination with fresh, whole, high quality ejaculates, which was done by placing a syringe at the external cervical os and gently expelling the inseminate in a pulsatile fashion to mimic coitus. The seminal pool remained in the proximal vagina throughout the procedure. The surgical approach utilized a small horizontal abdominal incision at the pubic hair line. Following visualization of the uterus and tubes, a small Kelly clamp was placed at the tubo-uterine junction, the isthmic-ampullary junction, and the ampullo-fimbrial junction of each tube. Salpingectomy was accomplished by placing multiple Kelly clamps beneath the junction of the tube and the broad ligament, initially severing the tube from the fimbriae ovaricae and then from the uterus at the serosal surface of the uterus. The remaining broad ligament was ligated around the separate clamp pedicles. The time of clamping was different for each tube in each subject to minimize differences between women in our results. Following excision, the fallopian tubes were divided into fimbrial, ampullary, and isthmic segments. These were opened and then vigorously rinsed in test tubes containing 1 ml. of serum. The tubal segments were removed and fixed for microscopic examination. The cervical mucus column was re- -«

3 Septem ber 1973 SPERM TRANSPORT IN WOMEN 657 moved after the salpingectomy procedure by the technic of Davajan and Kunitake,4 without contacting vaginal contents. In this fashion, cervical mucus present at the tip of the tubing represented that at the level of the internal cervical os, and that closer to the syringe represented mucus at the external os. An endometrial cavity washing was then performed utilizing Gravlee jet washes of 5 ml. of normal saline in 2.5-ml. aliquots, followed by dilatation and curettage. During the surgical procedure, one investigator evaluated the donor ejaculate used for insemination. Donors were healthy, presumably fertile, male volunteers with known high quality ejaculates who had not ejaculated for 72 hr. prior to the study. Ejaculates were obtained by masturbation and collected directly into a sterile bottle maintained at body temperature. The total ejaculate volume was measured following liquefaction and was inseminated within 30 min. after collection. A 0.5-ml. aliquot was immediately assessed for quality of sperm motility, density per milliliter, and sperm morphology, and the total number of sperm inseminated and the total number of motile sperm inseminated were calculated. Rinses of the tubal segments and the endometrial and curettage washings were kept carefully isolated from each other and evaluated for sperm content. Rinses were divided into 0.5-ml. portions and two drops of 1 % saponin were added to lyse red blood cells. The portions were then cytocentrifuged at 500 r.p.m. for 50 min. During this procedure, cellular material was deposited on glass slides in a 0.5-cm. circular area and the fluid was decanted onto filter paper. The slides were carefully labeled and stained with Giemsa according to the May-Greenwald technic. ll After drying, the slides were cover-slipped and scanned under phase-contrast 400x magnification. Any sperm identified were confirmed by examination at 1000x oil immersion magnification. The cervical mucus column was assessed for the presence of sperm at the level of the internal os by direct observation in the polyethylene tubing at 400x magnification. It was then dissolved in saturated fructose solution, and the concentration of sperm in the column was determined by a standard sperm-counting chamber technic. 12 Using these methods, 60% of known numbers of sperm added to segments of excised tubes are recovered from the tubal rinse before cytocentrifugation, as assessed by the counting chamber. Recovery of tubal sperm after cytocentrifugation and TABLE 1. Characteristics of Study Subjects Subject H A He L W G B Beau Age (yr.) Gravidity Last coitus (days pre operative) Sperm negative (days preoperative) Estradiol (pg./ml.) Surgery (cycle day) Spinnbarkheit (cm.) >8 >6 >5 >6 >8 >10 >10 >8 Fern Ovarian finding Corpus Follicle Corpus Follicle Follicle Follicle Follicle Follicle luteum luteum

4 658 SETILAGE ET AL. Vol. 24 staining is 1%. Sperm numbers reported are absolute sperm observed. Significance of difference in results was calculated by the Student t test. RESULTS Characterization of study subjects is presented in Table 1 and description of inseminations is presented in Table 2. No sperm were found in tubes removed before insemination or in tubes removed 2 min. after insemination. Sperm were present in the oviducts of 2 subjects within 5 min. following insemination and in the oviducts of all subjects studied at later times (Table 3). Additionally, sperm were distributed TABLE 2. Description of Inseminations Subject Total sperm Sperm Normal inseminated motility sperm x 10' % H 750, 65%; A %; He %; L %; W %; G %; B 59 60%; Beau %; Subject TABLE 3. Sperm Present in Oviducts Minutes Sperm present after insemini- Isth Am- Fimnation mus pulla bria Total B Beau B Beau He L He L A G W A H W H G throughout all segments of the oviducts. The total numbers of oviductal sperm found related directly to the numbers of sperm in the insemination (Fig. 1). As seen in Table 4, determination of the ratio of sperm in the oviduct to the sperm inseminated and to the motile sperm inseminated revealed a significant difference between the numbers found at 5 min. and those found at 10 min. (p < 0.05), and between those found at 10 min. and those found between 15 and 45 min. (p < 0.01). From 15 to 45 min. after insemination, the ratios remained constant, indicating no ffi 40 a:l ~ 35 ~ Z 30 ~ 25 w ~ _ > '.otal.perm (n' 12)... Meonforoll,n.. m;no'''ln,16} < ' '0'01 Sporm In'.} o MINUTES FIG. 1. Effect of inseminate quality on tubal sperm. TABLE 4. Ratio of Tubal Sperm to Inseminated Sperm Tubal: total sperm Tubal: motile sperm Minutes x 10' x 10' 5 1:126 1:82 5 1:155x=I:141 1:I08i = 1: :34 1: :24 i = 1:29 1:17 i = 1: :9 1:7 18 1:9 It = 1: 12 1:6 It = 1:7 18 1:15 1:8 30 1:21 1: :14 x = 1:15 1:8 i = 1:8 30 1:9 1:4 45 1:17 1: :11 i = 1: 14 1:8 i = 1:9

5 September 1973 SPERM TRANSPORT IN WOMEN 659 further increase in sperm number with increasing length of time from insemination (Fig. 2). Calculation of the percentage of sperm found in each segment for each studied oviduct, and then combination of the percentages for all oviducts studied, revealed that consistently the greatest numbers of sperm were present in the fimbria (Fig. 3). In only 1 subject were any sperm found in either endometrial or curettage washings. This sample was obtained 80 min. after insemination and consisted of 250 sperm. Sperm found in cervical mucus related directly to the sperm inseminated (Table 5). The relationship of these sperm to the motile sperm inseminated seems to be more consistent than to the total sperm inseminated. The ratio of cervical mucus sperm to inseminated sperm is constant from 15 to 80 min. By analyzing our over-all data we can find no relationship between cervical mucus and oviductal sperm to endometrial cavity sperm because of the absence of endometrial sperm. However, in the period of min. after insemination, one of every 14 million deposited sperm is in the oviduct, one of every 2000 deposited sperm is in the cervical mucus, and one of every 5000 sperm in the cervical mucus is in the oviduct. -0 1:160 ::'0 ~~ 1:140 2:E 1:120 LL.0.: O~ 1:100 V) 00 1:80 -w 4:..., 1:60 0.:«_ Total Inseminated Sperm... Motile Inseminated Sperm Z ~ 1:40 «:E :E ~ 1: 20 L...L..-=::::S;:::Jc:!:3:::;~t. o MINUTES FIG. 2. Relationship of tubal sperm to inseminated sperm :.e 0 60 :E 50 Q: w 0-40 Vl II D ISTHMUS AMPULLA LOCATION ~ < 15m,nu'., In'4) c=:j > 15m,nu'., In' 6) _ All tom (n'lo) fimbria FIG. 3. Tubal location of sperm. TABLE 5. Sperm Present in Cervical Mucus Total inseminated sperm Sperm Ratio to Ratio to Min motilutes Count total motile ity sperm sperm x 10' % x 10' x 10' x 10' :37 1: :74 1: :20 1: :25 1: :23 1: :34 1: :30 1: :13 1:8 DISCUSSION Early sperm transport utilizing a reproducible technic in normal midcycle women is described. The rapidity of sperm travel to the oviduct suggests either that sperm travel more rapidly in vivo than they do in vitro or that the female reproductive tract assists their passage. 2, 14 Recovery of more sperm from the fimbria than from other segments of the oviduct is counter to an assumption that sperm remain in the ampulla until fertilization occurs. It strongly supports, as does the constancy of sperm numbers found between 15 and 45 min. after insemination, a theory that sperm traverse the oviduct and are discharged into the peritoneal

6 660 SETTLAGE ET AL. Vol. 24 cavity.3, 16 If they remained in the oviduct, the numbers of recovered sperm would be expected to increase as the time from insemination increased. Such data may be recorded as longer times from insemination are studied, but they were not seen in this study. Surprisingly, it appears that the presence of sperm in the endometrial cavity is not a feature of early sperm transport in the human. J aszczak 19 described subsurface endometrial glands containing many sperm, with few sperm in the cavity of the nonhuman primate. Such might be the case in the human as well, serving as directing channels for sperm in transuterine migration. Total numbers of sperm in cervical mucus and the relationship of these to the insemination and the time from insemination have not been described previously. Beyond 15 min., we saw no real increase or decrease in numbers of sperm in cervical mucus. Several possibilities exist to explain this. The ascendency rate of sperm into the uterus and fallopian tubes may be relatively slight enough that we cannot yet determine a change, or the rate of vaginal sperm penetration into cervical mucus might equal the rate of sperm ascending higher in this early time period. The preliminary data presented here offer enticing possibilities regarding the phenomenon of sperm transport in the human female reproductive tract. Confirmation of observations, extension of data, and further characterization require additional study. SUMMARY A technic for assessing optimal sperm transport features in women is presented. Use of this technic has identified sperm in the oviduct within 5 min. from deposition in the proximal vagina. The numbers of sperm found in the oviduct were directly related to the numbers of sperm in the inseminate. Sperm were distributed throughout the oviduct and were in highest numbers in the fimbria. A constant level of sperm in the oviduct existed from 15 to 45 min. after insemination. Similar observations on sperm present in the endometrial cavity have revealed no sperm until 80 min. after insemination (in 1 subject). The total numbers of sperm present in cervical mucus are reported and correlated to the inseminate and the time following insemination. REFERENCES 1. MOYER, D. L., RIMDUSIT, S., AND MISHELL, D. L., JR. Sperm distribution and degradation in the human female reproductive tract. Obstet Gynec 35:831, SOBRERO, A. J. In Mechanisms Concerned with Conception, Hartman, C. G., Ed. Pergamon Press, New York, 1963, Chap. 4, p THIBAULT, C. Physiology and physiopathology of the fallopian tube. Int J Fertil 17:1, DAVAJAN, V., AND KUNITAKE, G. M. Fractional in vivo and in vitro examination of post-coital cervical mucus in the human. Fertil Steril 20:197, MACLEOD, J., MASTEN, F., SILBERMAN, C., AND SOBRERO, A. J. The post-coital and post-insemination cervical mucus and semen quality. Fertil Steril 10:41, PERIOFF, W. H., AND STEINBERGER, E. In-vitro penetration of cervical mucus by spermatozoa. Fertil Steril 14:231, SCHWARZ, R. Some factors modifying sperm progression. Fertil Steril 2:15, DEBoER, C. H. Transport of particulate matter through the human female genital tract. J Reprod Fertil 28:295, EGLI, G. E., AND NEWTON, M. The transport of carbon particles in the human female reproductive tract. Fertil Steril 12:151, BEHRMAN, S., AND KISTNER, R. Progress in Infertility. Little, Boston, MANN, T. Biochemistry of Semen and of the Rep.,.oductive Tract. Wiley, New York, SAGA, M., NAKAMURA, R., AND SETTLAGE, D. Unpublished data. 13. RUBENSTEIN, B. B., STRAUSS, H., LAZARUS, M. L., AND HANKIN, H. Sperm survival in woman. Fertil Steril 2:15, BROWN, R. L. Rate of transport of spermia in human uterus and tubes. Amer J Obtet Gynec 47:407, 1944.

7 Septem ber 1973 SPERM TRANSPORT IN WOMEN MOGHISSI, K. S. Human and bovine sperm migration. Fertil Steril 19:118, HORNE, H. H., JR., AND THIBAULT, J. P. Sperm migration through the human female reproductive tract. Fertil Steril 13:135, AHLGREN, M. "Number of Spermatozoa in the Ampulla of the Human Fallopian Tube" (abstr. 41). In Seventh World Congress for Fertility and Sterility, Tokyo, FAUNDES, A., CROXATTO, H., MEDEL, M., VERA, C., AVENANS, S., AND ANSELMO, J. "Sperm Migration in the Female Genital Tract" (abstr. 39). In Seventh World Congress for Fertility and Sterility, Tokyo, JASZCZAK, S. "Migration of Sperm in the Cervix and Uterus of Non-human Primates from Cervical Mucus in Human Reproduction." In World Health Organization Colloquium, Geneva, p.33.