the Female Reproductive Tract

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1 BIOLOGY OF REPRODUCTION 3, (98) Assessment of Human perm Function After Recovery from the Female Reproductive Tract JOHN E. GOULD, JAME W. OVERTREET and FREDRICK W. HANON Division of Reproductive Biology and Medicine Department of Obstetrics and Gynecology chool of Medicine University of California Davis, California 9566 ABTRACT The physiology and fertile life of human spermatozoa in the female reproductive tract have received little previous attention. A technique was developed for recovering spermatozoa from human cervical mucus at various intervals after artificial insemination. The functions of these cells as measured by penetration of the human zona pellucida and fusion with the zona-free hamster oocytes were examined. Penetration into the zona pellucida was consistently observed when sperm were recovered from to 8 h after insemination. Penetration through the zona into the perivitelline space (PV) was seen from to 7 h after insemination. Fusion of human sperm with zonafree hamster oocytes was observed from to 8 h after insemination. Motile sperm were recovered and h after insemination with swimming speeds comparable to freshly capacitated spermatozoa. Concentrations of recovered sperm at these longer intervals from insemination were insufficient for sperm-oocyte assays. These studies demonstrate that human spermatozoa aged in vivo may be recovered from cervical mucus for physiologic study, and suggest that the fertile life of human sperm may be 8 h or more. INTRODUCTION The duration of human sperm fertilizing capacity in the female is unknown, and human sperm physiology during aging in the female reproductive tract has not previously been studied. Previous estimates of the maximal fertile life (-8 h: Young, 96; Blandau, 969) and motile life (8 h-7 days: Young, 96; Perloff and teinberger, 96) of human spermatozoa have been based on the recovery of motile sperm from the cervical mucus, uterus, oviducts and peritoneal fluid. The estimates of fertile life, however, are purely conjectural and are based on the widely accepted hypothesis that loss of sperm fertility precedes loss of motility in most mammalian species. These issues are further complicated by evidence that loss of sperm fertility may be preceded by a phase of senescence during which sperm may give rise to abnormal embryos (Austin, 975), which may or may not develop to a stage of recognizable pregnancy. Accepted August 6, 98. Received May 3, 98. Reprint requests. Reliable estimates of the duration of the sperm fertility can be made in some animal species; for example, the guinea pig ovulates h after the onset of heat, and the rabbit ovulates h after copulation. Delayed insemination in the former or sterile copulation followed by insemination in the latter permit accurate estimates of the fertile life of spermatozoa. uch estimates range from 6-3 h for species such as rabbits, mice, guinea pigs and rats, to months and years for bats and snakes, respectively (Young, 96; Austin, 975). For humans, a species with continuous female sexual receptivity and variable times of ovulation, reliable estimates of the duration of sperm fertility are not available. Natural family planning (rhythm) methods of contraception are based on certain assumptions about the time of ovulation as well as sperm and oocyte longevity. It is generally believed that spermatozoa are capable of fertilizing an oocyte for 8-7 h (DHH Publication No. 8-56). This assumption, together with those which define the time of ovulation and oocyte survival, form the basis for recommended periods of abstinence and safe intercourse. There are no reliable scientific data which 888 on 6 November 7

2 HUMAN PERM RECOVERED FROM CERVICAL MUCU 889 define the fertile life of human spermatozoa. In view of the worldwide use of natural family planning it is desirable to determine the duration of human sperm fertility in the female with greater accuracy. The relationship between aged gametes and adverse pregnancy outcome in women is unresolved, but a relationship between infrequent coitus and fetal chromosomal aberrations has been postulated (German, 968). It is therefore important to study the biology of aging human gametes. In this paper, we introduce a technique for recovering human spermatozoa which have aged within the female tract and we describe experiments with these gametes which examine some of the sperm functions involved in fertilization. Preparation of Oocytes MATERIAL AND METHOD Human oocytes were recovered from ovarian tissue, stored at -8#{76}C and prepared for incubation as previously described (Overstreet et al., 98). Briefly, this method involves mincing whole human ovaries and passing the tissue through a series of grids; oocytes will pass through the large grid and collect on the fine grid. The oocytes are frozen and stored in phosphatebuffered saline (PB) containing M dimethyl sulfoxide (DMO). These oocytes were never viable and could not be fertilized in vitro; they were used to assess sperm interaction with the zona pellucida (Gould et al., 983). Zona-free hamster oocytes were used to assess sperm fusion with the oolemma and were obtained by standard methods for superovulation, ovum recovery and removal of the zona pellucida (Yanagimachi et al., 976). Recovery of Cervical Mucus Cervical mucus was collected from women who were patients in our artificial insemination program; all had infertile husbands and were undergoing artificial insemination by donor (AID). The mucus was collected at intervals after insemination in a polyethylene catheter as previously described (Katz et al., 98). In this technique a sample of cervical mucus is aspirated into a -cm length of polyethylene tubing attached to a syringe. During aspiration the catheter tip is inserted to cm into the cervical canal. In the present experiments the mucus was then screened by examining a small amount between a slide and coverslip to ensure that viable cells were present. The number of motile spermatozoa per microscope field was estimated (Hanson et al., 98), the interval since insemination was noted, and the remaining mucus was immediately processed for recovery of sperm cells. perm Recovery from Cervical Mucus The procedure for recovering sperm from human cervical mucus is depicted in Fig.. Mucus was expelled Expel Mucus into BWW medium Mucus in Polyethylene Tubing Mucus Medium lncubatel hr at 37#{76}C to allow sperm to enter BWW medium Remove and gloss bead filter BWW medium now WW containing sperm Glass beods 6 medium with reccvere sperm Centrifuge eluted BWW medium PERM RECOVERY FROM HUMAN CERVICAL MUCU BWW medium _- permatozoa Human oocytes Incubate hours oil at 37#{76}C oil under 5% Co Hmster perm uspension vitelli Add #{3}ocytes and Place suspension in flat capillary vitelli to tube tube I [L) :Oil] perm suspension 3l) Remove super - notont, leaving spermatozoa in 3 pi BWW perm uspension FIG.. perm recovery from human cervical mucus. on 6 November 7

3 89 GOULD ET AL from the polyethylene collection tubing using a loo-ml micropipette plunger (Drummond ci. Co., Broomall, PA) (cf., Katz et al., 98). The mucus was expelled directly into 3 to 5 ml of BWW medium (Yanagimachi et al., 979) supplemented with 35 mg/mi human serum albumin (HA, Fraction V, igma Chemical Co., t. Louis, MO). The mucus was incubated for 5 mm to h at 37#{76}Cunder 5% CO3 in air, allowing spermatozoa to pass from the hydrated mucus into the BWW medium. The mucus was removed at the end of the incubation with a Pasteur pipette. The sperm suspension left behind was either centrifuged immediately at 5 X g for 6 mm or first passed through a glass bead column cm long and 3 mm in diameter (VWR cientific,.5- to.3-mm beads) and then centrifuged. The glass bead column was introduced into the protocol midway through the experiments and was used in 5 of the 7 experiments with oocytes. It appeared to be useful in removing dead sperm and cellular debris from the sperm suspension. After centrifugation of the sperm suspension the supernatant was removed until -5 sl of medium remained in the bottom of the centrifuge tube. By gentle mixing, a final volume of sperm suspension was prepared. Twenty to 5 M of the suspension was injected with a micropipette into a -Mm flat glass capillary tube (Vitro Dynamics, Rockaway, NJ). An oil seal was created at both ends of the fiat tube by injecting mineral oil (E. R. quibb, Princeton, NJ) with a 3-gauge needle and syringe (cf., Overstreet et al., 98). Assessment of perm Function One to human oocytes and/or 6 to 3 zona-free hamster oocytes (vitelli) were injected into the flat tube with a flame-drawn micropipette. Videomicrographic recordings of sperm movement in the flat tube were made immediately for subsequent assessment of percent motility and swimming speed, using a video camera mounted on a microscope and a video cassette recorder, as previously described (Katz and Overstreet, 98). Recordings were made close to the upper and lower inner surfaces of the flat tube, since most spermatozoa swam along those planes. The concentration of spermatozoa in the flat tube was estimated at the time of videomicrographic recording by counting the number of sperm throughout the depth of a X field ( \.5 Ml); this number was applied to a standard curve generated by the same counting procedure using sperm suspensions of known concentrations. Incubation of the flat tube containing sperm and oocytes was carried out over h at 37#{76}Cunder 5% CO3 in air. The oocytes and vitelli were recovered with a flane-drawn micropipette. Human oocytes were mounted between a glass slide and coverslip and were rolled to identify spermatozoa in the zona pellucida and perivitelline space (Overstreet and -lembree, 976). Hamster vitelli were evaluated for attached sperm as well as decondensing sperm heads within the ooplasm by compression between a coverslip and slide (Blazak et al., 98). REULT The experiments were conducted over a 3-mo period. Approximately 6 human cervical mucus samples were examined after artificial insemination to obtain 6 samples suitable for sperm recovery. The initial estimate of sperm concentration in those 6 samples ranged from one cell per x field to cells per x field. The volume of the mucus samples ranged from 3-8 p. Postinsemination intervals studied ranged from - h. After recovery from the cervical mucus, the concentration of sperm in the final suspension ranged from less than i#{8}per ml to 5 x 6/ml; the recovered cells were essentially % motile. During initial observations, it became clear that a minimal sperm concentration was required for in vitro penetration of the human zona pellucida and hamster vitellus. We never observed penetration into the human zona pellucida with sperm concentrations less than 6 x j sperm/ml, and spermatozoa were never observed in the perivitelline space (PV) when the sperm concentration was less than i#{8} sperm/mi (Fig. ). We did not observe sperm fusion with the hamster vitellus when fewer than sperm/mi were present (Fig. ). When this concentration dependence became apparent, spermatozoa from of the 6 mucus samples were not exposed to oocytes because the concentrations of recovered sperm were thought to be inadequate for interpretation. Thus, sperm suspensions recovered from 7 mucus samples were used for incubation with oocytes in these experiments. Because of the logistical difficulties of coordinating hamster egg recovery and cervical mucus aspiration, human and hamster oocytes could not be tested in every experiment. pecifically, 3 mucus samples were used to obtain spermatozoa for testing with human oocytes and mucus samples produced sperm for testing with hamster vitelli; 6 mucus sampies were used with both gametes. The data for all experiments are summarized in Fig., irrespective of the time from insemination to mucus recovery. The figure indicates the results for 7 mucus samples, each symbol representing one experiment (one mucus sample per experiment). A total of 99 human oocytes were used in 3 of these experiments. Of 9 oocytes examined with recovered sperm concentrations 6 x only failed to show some degree of zona penetration. ince the positive symbols on Fig. indicate at least one successful penetration, it follows that unpenetrated oocytes were observed in experiments which included at least one other oocyte that was successfully penetrated. Finally, the on 6 November 7

4 HUMAN PERM RECOVERED FROM CERVICAL MUCU 89 #{9}. ZONA PENETRATION Positive Negative -I-. #{9}... #{9} #{9}#{9}#{9} #{9} #{9} #{9}5.... #{9} -F pv* PENETRATION Positive Negative : #{9}5 #{9}#{9} #{9} #{9}.. #{9}. 55 #{9} #{9}.. -- VITELLINE Positive FUION Negative. -I I. I #{9}#{9}#{9} PERM CONCENTRATION i l I I 3/mI /mi 5/mI 6/mI 7/mI FIG.. ummary of results for all intervals after insemination. Each symbol represents the results for one mucus sample. The concentration of spermatozoa recovered from each sample is shown on the bottom scale. *pvspe,jvitelline space. discrepancy between the number of symbols for zona and PV penetration in Fig. reflects the fact that all human oocytes have a zona pdlucida, but not all have a clearly defined PV. Thus, an oocyte was said to be negative for PV penetration only if it had a PV that could be evaluated. The ability of human spermatozoa to penetrate the human zona pellucida after different periods of residence in the female reproductive tract is shown in Table. The data in Table for zona penetration are based only on those experiments in which the concentration of recovered sperm was 6 x /ml, below which zona penetration was never seen. imilarly, the data for sperm penetration in the PV includes only experiments with concentrations of recovered sperm i /ml, below which PV penetration was never seen. Penetration of the zona pellucida was observed with sperm populations recovered as long as 8 h after insemination, and full penetration through the zona and into the PV was observed with populations recovered 7 h after insemination. Penetration TABLE. The ability of human spermatozoa to penetrate the human zona pellucida after different periods of residence in the female reproductive tract. Hours af ter artif icial insemination Totals No. mucus samples studied No. human oocytesa with zona sperm (96%) Total no. oocytes examinedb No. human oocytes with PVc sperm (9%) Total no. oocytes examinedd apreviously frozen nonviable human oocytes. blncludes data for concentrations of recovered sperm 6 X io fml. cpv it Hi space. dlncludes data for concentrations of recovered sperm #{76}/mI. on 6 November 7

5 89 GOULD ET AL. of the zona pellucida was consistently observed when sperm numbers were sufficient, whereas sperm entry into the PV was more variable. There is no indication of attenuation of zona penetrating ability during 8 h of sperm residence in the female tract. Observations of sperm fusion with hamster vitelli are summarized in Fig., across all postinsemination intervals. Results for mucus samples are shown; sperm incorporation into the zona-free oocytes was not observed when the sperm concentration was less than i#{5} sperm/ml. Above this threshold, penetration rates were inconsistent until sperm concentrations reached x 6/ml, when 35 of 9 vitelli were penetrated. The ability of human spermatozoa to fuse with the hamster vitelli after different intervals of residence in the female tract is also shown in Table (only experiments with sperm concentrations > / ml included). Fusion with hamster vitelli was observed with sperm populations recovered up to 8 h after artificial insemination, but only three attempts were made at intervals longer than 8 h. permatozoa were recovered from cervical mucus at 96,, and h after insemination, but in all experiments the recovered sperm concentration was </ml. No oocytes were tested in view of this low concentration, but sperm numbers were often sufficient to measure swimming speeds. The mean swimming speeds (± EM) of these spermatozoa ranged from 85. ± 5.6 pm/sec at 7 h to 63.7 ±.7 pm/sec at h (N=5). DICUION The majority of human spermatozoa which reside in the cervical mucus are motile and our recovery methods provide a population of vigorous sperm for laboratory study. We do not know exactly how spermatozoa leave the mucus after recovery. It is likely that mucus hydration occurs following a Donnan equilibrium (Tam and Verdugo, 98). As the entangled networks of mucin swell with medium, it is possible that spermatozoa encounter aqueous channels through which they can leave the mucus microstructure. These studies demonstrate that human spermatozoa may be recovered from cervical mucus, providing a method for study of physiology of human sperm following prolonged interaction with the female reproductive tract. Human spermatozoa recovered from the cervical canal appear to have achieved at least a state of partial capacitation in vivo. perm recovered from the mucus h following insemination were able to penetrate the human zona pellucida and to fuse with the zona-free hamster oocytes, physiological events which require both capacitation and the acrosome reaction (Yanagimachi et al., 976; Barros et al., 979). It could be argued that the in vitro conditions utilized in these experiments could have induced capacitation during the period of sperm oocyte incubation. However, other studies we have carried out support the notion that these spermatozoa were capacitated before recovery from the mucus (Lambert et al., 98). We have also developed an in vitro capacitation system in which spermatozoa are separated from the seminal plasma by migration through a column of human cervical mucus, rather than by conventional washing and centrifugation techniques. Our initial experiments with the TABLE. The ability of human spermatozoa to fuse with zona-free hamster oocytes after different periods of residence in the female reproductive tract.a Hours af ter artif icial insemination Totals No. mucus samples 8 7 No. hamster vitelli penetrated 9 6(6%) Total no. vitelli examined No. expts. with vitellus penetrated 7 Total no. experiments 8 8 alncludes data for concentrations of recovered sperm >5/ml. on 6 November 7

6 HUMAN PERM RECOVERED FROM CERVICAL MUCU 893 system demonstrated a rapid sperm penetration of the zona pellucida (Overstreet et al., 98), a characteristic which we have now observed with sperm populations capacitated in vivo. The physiological state of the in vitro mucuscapacitated sperm appears to be similar in other respects to spermatozoa which have been capacitated in the human cervical canal. The concentration of spermatozoa required for zona penetration is similar in the two systems (compare Fig. with Fig. 3 of Overstreet et al., 98). Furthermore, exposure to cervical mucus in vitro appears to alter the interaction of capacitated human spermatozoa with the hamster vitelli (Barros et al., 983). In comparison with sperm populations capacitated by traditional washing procedures, mucus-capacitated sperm bind less avidly to the surface of the hamster vitellus and fewer vitelli are penetrated (Gould et al., 98). This diminished capacity for interaction with the hamster vitellus also appears to be a characteristic of human sperm capacitated in vivo, since the attachment and penetration rates observed in these experiments closely resemble those of in vitro mucus-capacitated sperm as opposed to that of washed sperm (unpublished observations). Previous estimates of the duration of sperm fertility in the human female tract are speculative. In 95, Rubenstein et al., recovered sperm from the uterine cervix, fundus and tubes of 5 hysterectomy patients. They found motile sperm at the longest postinsemination intervals examined (5 h), although the authors conceded that fertility may not correlate with sperm motility. In 958, Home and Audet recovered motile sperm from the peritoneal fluid 36 h after coitus in 3 infertile women. Although observations on sperm motility from the late 8 s suggested that spermatozoa might survive in the Fallopian tubes for several weeks after coitus (Hartmann, 93), more recent data suggest that human sperm motility in the female may be preserved for no longer than wk (Perloff and teinberger, 96). Other estimates of sperm fertilizing ability rely on indirect statistical methods which assess the risk of conception as a function of coitus and changes in basal body temperature (Barrett and Marshall, 969), or on incidental findings in hormonal studies of ovulation and pregnancy (Thomas et al., 973). The former estimate is days, the latter 5 days. Other references which are often cited on the fertile life of human sperm are conjectural (Pouchet, 87; Farris, 956), and existing data are inadequate for any conclusions to be drawn (Austin, 975). Our observations demonstrate that spermatozoa which have resided in the female tract for as long as 8 h retain the ability to undergo the acrosome reaction and can penetrate into the human zona peliucida; sperm entry into the PV was demonstrated up to 7 h following insemination. The spermatozoa recovered at 8 h did not reach the PV, but the interpretation of this single experiment is not certain. permatozoa as old as 8 h could be shown to incorporate into the hamster ooplasm. These are probably underestimates of the duration of these functions since negative results at longer intervals could also be attributed to an inadequate number of recovered sperm. Furthermore, swimming speeds of spermatozoa recovered 7 to h after insemination are comparable to swimming speeds of freshly capacitated spermatozoa (Blazak et al., 98). Our findings support the notion that human sperm fertilizing capacity may persist in the female tract for 8 h or longer. However, the senescense of mammalian spermatozoa may be gradual, with the spermaotoza losing the capacity to initiate normal embryonic development before losing the ability to fertilize (Austin, 975). These experiments demonstrate an approach which may be useful for study of the human sperm capacitation phenomena in vivo. ACKNOWLEDGMENT This work was supported by NIH grant HD59 and a Research Career Development Award to J.W.O. (HD). The authors gratefully acknowledge the assistance of Ms. Pat Blondheim in preparing this manuscript. REFERENCE Austin, C. R. (975). perm fertility, viability and persistence in the female tract. J. Reprod. Fertil. uppl. : Barrett, J. C. and Marshall, J. (969). The risk of conception on different days of the menstrual cycle. Popul. tud. 3:55-6. Barros, C., Gonzalez, J., Herrera, E. and Bustos-Obregon, E. (979). Human sperm penetration into zona-free hamster ocoytes as a test to evaluate the sperm fertilizing ability. Andrologia (3), 97-. Barros, C., Vigil, P., Herrera, E., Perez, A., Guadarrama, A. and Bustos-Obregon, E. (983). In vitro interaction betwen human spermatozoa and human cervical mucus. Biol. Cell. 7(),3-9. Blandau, R. J. (969). Gamete transport-comparative aspects. In: The Mammalian Oviduct (E..E. on 6 November 7

7 89 GOULD ET AL Hafez and R. J. Blandau, eds.). University of Chicago Press, Chicago, p.. Blazak, W. F., Overstreet, J. W., Katz, D. F. and Hanson, F. W. (98). A competitive in vitro assay of human sperm fertilizing ability utilizing contrasting fluorescent sperm markers. J. Androl. 3(3):65-7. DHH Publication No Natural Family Planning. Department of Health and Human ervices, Health ervices Administration, United tates Government. Farris, E. J. (956). Human Ovulation and Fertility. J. B. Lippincott, Philadelphia, p.. German, J. (968). Mongolism, delayed fertilization and human sexual behavior. Nature 7:56. Gould, J. E., Overstreet, J. W. and Hanson, F. W. (98). Penetration of zona-free hamster ova as an assay of human sperm function. Clin. Res. 9: 7A. Gould, J. E., Overstreet, J. W., Yanagimachi, H., Yanagimachi, R., Katz, D. F. and Hanson, F. W. (983). What functions of the sperm cell are measured by in vitro fertilization of zona-free hamster eggs? Fertil. teril. (3):3-35. Hanson, F. W., Overstreet, J. W. and Katz, D. F. (98). A study of the relationship of motile sperm numbers in cervical mucus 8 h after artificial insemination with subsequent fertility. Am. J. Obstet. Gynecol. 3():85-9. Hartman, C. G. (93). Ovulation and the transport and viability of ova and sperm in the female genital tract. In: ex and Internal ecretions (E. Allen, ed.). Williams & Wilkins Co., Baltimore, p. 7. Home, H. W. and Audet, C. (958). pider cells, a new inhabitant of peritoneal fluid. Obstet. Gynecol. ():-3. Katz, D. F. and Overstreet, J. W. (98). perm motility assessment by videomicrography. Fertil. teril. 35(): Katz, D. F., Overstreet, J. W. and Hanson, F. W. (98). A new quantitative test for sperm penetration into cervical mucus. Fertil. teril. 33(): Lambert, H., Overstreet, J. W., Morales, P., Hanson, F. W. and Yanagimachi, It (98). perm capacitation in the human female reproductive tract. Fertil. teril. (in press). Overstreet, J. W. and Hembree, W. C. (976). Penetration of the zona pellucida of nonliving human oocytes by human spermatozoa in vitro. Fertil. teril. 7(7): Overstreet, J. W., Gould, J. E., Katz, D. F. and Hanson, F. W. (98). In vitro capacitation of human spermatozoa after passage through a column of cervical mucus. Fertil. teril. 3(6): Perloff, W. H. and teinberger, E. (96). In vivo survival of spermatozoa in cervical mucus. Am. J. Obstet. Gynecol. 88():39-. Pouchet, F. A. (87). Theorie positive de l ovulation spontanee et de Ia fecondation des mammiferes el l espece humaine basee sur l observation de toute Ia serie animale. Balliere, Paris. Rubenstein, B. B., trauss, H., Lazarus, M. L. and Hankin, H. (95). perm survival in women: motile sperm in the fundus and tubes of surgical cases. Fertii. teril. ():5-9. Tam, P. Y. and Verdugo, P. (98). Control of mucus hydration as a Donnan equilibrium process. Nature 9:3-3. Thomas, K., Dc Hertogh, R., Pizarro, M., Van Exter, C. and Ferin, J. (973). Plasma LH-HCG, 7j3-estradiol, estrone and progesterone monitoring around ovulation and subsequent nidation. Int. J. Fertil. 8: Yanagimachi, It, Yanagimachi, H. and Rogers, B. J. (976). The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Biol. Reprod. 5 :7-76. Yanagimachi, R., Lopata, A., Odom, C. B., Bronson, R. A., Mahi, C. A. and Nicolson, G. L. (979). Retention of characteristics of zona peilucida in highly concentrated salt solution: the use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil. teril. 3 : Young, W. C. (96). ex and Internal ecretions. Williams & Wilkins, Baltimore, p on 6 November 7