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In-vitro Penetration of Cervical Mucus by Spermatozoa
In-vitro Penetration of Cervical Mucus by Spermatozoa WILLIAM H. PERLOFF, M.D., and EMIL STEINBERGER, M.D.
KrusTELLER, 1 in 1896, was the first to suggest that spermatozoa actively traverse the cervical canal by virtue of their own motility rather than passively by action of the female genital tract during orgasm. Subsequently, Strassmann and Seeligmann confirmed these conclusions, the latter postulating a chemotactic mechanism. In 1932, Miller and Kurzrok re-examined seminal fluid-cervical mucus relationships and described a technic for measuring the ability of spermatozoa to penetrate cervical mucus from seminal fluid. As described by them, a drop of semen was placed near a drop of cervical mucus on a clean, dry, glass slide and the margins of the drops were approximated by the weight of a cover slip, which was allowed to fall squarely on them. The contact zone was examined' microscopically under high dry power and progress of spermatozoa into cervical mucus was noted. Under normal circumstances, these authors reported, sperm~tozoa penetrated cervical mucus in the form of a pyramid with the apex pointing into the mucus, a formation they termed "phalanx." The phalanx gradually increased in size and depth of penetration. The advancing spearhead of spermatozoa split, and each column advanced separately in different directions with eventual dissemination of spermatozoa throughout the substance of the cervical mucus. In this report we present observations made from application of phase microscopy to the Miller-Kurzrok procedure.
,
METHOD Twenty regularly menstruating and normally ovulating women were studied. Cervical mucus was removed from the cervical canal after cleaning the external os at the time of ovulation. The seminal fluid-cervical mucus system was prepared as described by Miller and Kurzrok and specimens were From the Division of Endocrinology and Human Reproduction, Research Laboratories, Albert Einstein Medical Center, Philadelphia, Pa. This paper was presented before the American Society for the Study of Sterility, 18th Annual Meeting, Chicago, Ill., Mar. 30--Apr. 1, 1962.
231
232
PERLOFF
& STEINBERGER
FERTILITY
& STERILITY
studied and photographed with the Zeiss "Photoscope," employing phasecontrast optics. RESULTS
In most preparations one could visualize phase lines between cervical mucus and seminal fluid immediately following approximation of the two fluids (Fig. 1). Shortly thereafter, spermatozoa-filled phalanges could be observed. Phalanges, which uniformly projected into cervical mucus and never in reverse direction, soon developed marked degrees of arborization (Fig. 2), the terminal aspects of which consisted of canals through which only single spermatozoa or at most 2 spermatozoa abreast were able to pass (Fig. 3). Canals appeared to be long finger-like projections of seminal fluid into cervical mucus as evidenced by the ability of spermatozoa which had broken through into the mucus from other areas to pass immediately over andjor under the canals. As a single spermatozoon progressed along a canal, it was soon followed by a column of other spermatozoa. When the leader spermatozoon was blocked in its advance, those which followed ceased their forward motion. With resumption of forward progress by the leader spermatozoon, followers would likewise advance. Spermatozoa were observed to penetrate the substance of cervical mucus, singly or in groups, from the ends or sides of canals as well as from all phase lines between the two fluids. When one spermatozoon traversed a phase line, others in close proximity slipped through the same "opening." It was noted frequently that spermatozoa progressing actively within the substance of cervical mucus stopped abruptly as if they had encountered an impediment which prevented forward progress. Single or multiple spermatozoa so affected increased their activitj; thrashing about as though trying to break through this obstacle. After variable periods of time, either singly or together, they overcame the barrier and were able to progress further into the substance of the mucus. Careful observation of these areas by phase microscopy often revealed what appeared to be intramucus phase lines (Fig. 4). Having confirmed Miller and Kurzrok's thesis of phalangeal formation, we attempted to ascertain whether this was a function of spermatozoal activity, as they postulated, or an inherent characteristic of the two fluids predicated on their physicochemical properties. Accordingly, we introduced azoospermic specimens into the test system and noted development o.f phases and phalanges despite absence of spermatozoa in seminal fluid. These formations were also seen when azoospermic seminal fluid was tested against ovulatory cervical mucus containing large numbers of motile spermatozoa from insemination performed 24 hr. previously (Fig. 5). In such instances spermatozoa were seen to pass freely through phase lines from cervical mucus into azoospermic seminal fluid (Fig. 6). To evaluate further
VoL. 14, No. 2, 1963
SPERM AND CERVICAL
Mucus
233
I
Fig. 1. Phase-line formation at contact zone between seminal fluid and cervical mucus. A, Seminal fluid (streaked appearance of spermatozoa photographed in rapid motion). B, Cervical mucus, arrow pointing to phase line with early phalanx formation. Fig. 2. Arborization of phalanges in cervical mucus. A, Seminal fluid. B, Cervical mucus, arrow pointing to arborization of phalanges. Fig. 3; A, Seminal fluid. B, Cervical mucus, arrow pointing to a "canal" containing spermatozoa. Fig. 4, Cervical mucus 24 hr. after insemination showing intramucus phase lines.
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PERLOFF
& STEINBERGER
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& STERILITY
Fig. 6. Cervical mucus 24 hr. after insemination forming phase lines and phalanges with azoospermic seminal fluid. A, Seminal fluid. B, Cervical mucus, arrow pointing to early phalanx formation. Fig. 6. Penetration of spermatozoa from cervix mucus into seminal fluid. A, Seminal fluid. B, Cervical mucus, arrow c pointing to sperm moving across phase line from cervical mucus into seminal fluid, arrow d pointing toward phase line. Fig. 7. Phalanx formation between cervical mucus and azoospermic seminal fluid containing India ink. A, Seminal fluid. B, Cervical mucus, arrow pointing to phase line. Fig. 8. Phalanx formation between azoospermic India ink containing seminal fluid and cervical mucus. A, Seminal fluid. B, Cervical mucus, arrow pointing toward India-ink particles in
phalanx.
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235
the role of spermatozoa in phalanges and canal formation, the following experiment was conducted. Azoospermic seminal fluid was mixed with a small amount of India ink, and phase formation with ovulatory mucus was observed (Fig. 7). Phalanges and canals developed readily; India-ink particles penetrated into the canals (Fig. 8) and were also able to traverse phase lines into the cervical mucus. COMMENT
In this communication we have presented observations which confirm Miller and Kurzrok' s concept of microscopically demonstrable phase and phalanx formation at contact zones between ovulatory cervical mucus and seminal fluid. Whereas these authors ascribed formation of phalanges primarily to directed spermatozoal activity, we have provided evidence that phalanges develop not only at the boundary between cervical mucus and normal seminal fluid, but also between cervical mucus and azoospermic seminal fluid, indicating that participation of spermatozoa is not essential. Miller and Kurzrok reported spermatozoal passage from phalanges into cervical mucus. We have seen spermatozoa pass readily into cervical mucus from seminal fluid at any point of contact regardless of presence of phalanges. Consequently, phalangeal formation does not seem to be essential for spermatozoal penetration of cervical mucus. Inert (India-ink) particles suspended in azoospermic seminal fluid pass freely into phalanges and probably are transferred passively across all contact zones between seminal fluid and cervical mucus. Motility, therefore, does not seem to be an absolute prerequisite for penetration of cervical mucus, nor for formation of phalanges. Miller and Kurzrok and others3 • 4 have postulated a "chemotactic" attraction of cervical mucus for spermatozoa. Employing a system composed of spermatozoa containing cervical mucus from insemination 24 hr. previously, and azoospermic seminal fluid, we observed free passage of spermatozoa across contact zones from cervical mucus into seminal fluid. In these preparations, phalanges which formed at contact zones between the two fluids were directed from seminal fluid into cervical mucus, despite the fact that spermatozoa were traveling in reverse direction, lending further support to the suggestion that phalanges are not formed by spermatozoal activity. A chemotactic attraction by cervical mucus for spermatozoa is difficult to envision in view of these observations. In certain instances correlation between in-vitro and in-vivo penetration of cervical mucus by spermatozoa was not good, a phenomenon which requires explanation and is being investigated. Immediately following sexual intercourse, most of the ejaculate flows out
236
PERLOFF & STEINBERGER
FERTILITY & STERILITY
of the vagina, ·and .only a relatively small amount remains in contact with cervical mucus. In view of the relative slowness of cervical-mucus penetration py spermatozoa and rapidity with which spermatozoa are collected in the phalanges and canals, and in view of the tremendous increase in contact surface area occasioned by these formations, one cannot help but speculate as to the following sequence of events: 1. Approximation of ejaculate to cervical mucus 2. Formation of phalanges and canals with consequent trapping of a large number of spermatozoa 3. Gradual penetration of spermatozoa into cervical mucus from these repositories. CONCLUSIONS
When ovulatory cervical mucus and seminal fluid are placed in apposition in vitro, phase lines are formed from which phalanges and canals soon project into cervical mucus. This phenomenon does not depend upon the presence of spermatozoa. Many spermatozoa are trapped quickly in these repositories, from which they gradually penetrate cervical mucus. It is likely that phalanges and canals, in addition to pedorming a storage function, increase the sudace area between the two fluids which facilitate spermatozoal penetration of cervical mucus. Albert Einstein Medical Center York and Tabor Rds. Philadelphia 41, Pa.
REFERENCES I. KmsTELLER, S. Beitrage zu den Bedingungen der Conception. Berl. Klin. W chnschr. 8:315, 1871. 2. MILLER, E. G., JR., and KURZROK, R. Biochemical studies of human semen. III. Factors affecting migration of sperm through the cervix. Am. ]. Obst. & Gynec. 24:19, 1932. 3. SEELIGMANN, L. Weitere Mitteilungen vur Behandlung der Sterilitas Matrimonii. Zentralbl. f. Gyniik. 20:429, 1896. 4. STRASSMANN, P. "Vordringen der Spermien," in Winkel's Handbuch der Geburtschilfe, vol. 1, Part 1, Wiesbaden, 1903, p. 154.
KrusTELLER, 1 in 1896, was the first to suggest that spermatozoa actively traverse the cervical canal by virtue of their own motility rather than passively by action of the female genital tract during orgasm. Subsequently, Strassmann and Seeligmann confirmed these conclusions, the latter postulating a chemotactic mechanism. In 1932, Miller and Kurzrok re-examined seminal fluid-cervical mucus relationships and described a technic for measuring the ability of spermatozoa to penetrate cervical mucus from seminal fluid. As described by them, a drop of semen was placed near a drop of cervical mucus on a clean, dry, glass slide and the margins of the drops were approximated by the weight of a cover slip, which was allowed to fall squarely on them. The contact zone was examined' microscopically under high dry power and progress of spermatozoa into cervical mucus was noted. Under normal circumstances, these authors reported, sperm~tozoa penetrated cervical mucus in the form of a pyramid with the apex pointing into the mucus, a formation they termed "phalanx." The phalanx gradually increased in size and depth of penetration. The advancing spearhead of spermatozoa split, and each column advanced separately in different directions with eventual dissemination of spermatozoa throughout the substance of the cervical mucus. In this report we present observations made from application of phase microscopy to the Miller-Kurzrok procedure.
,
METHOD Twenty regularly menstruating and normally ovulating women were studied. Cervical mucus was removed from the cervical canal after cleaning the external os at the time of ovulation. The seminal fluid-cervical mucus system was prepared as described by Miller and Kurzrok and specimens were From the Division of Endocrinology and Human Reproduction, Research Laboratories, Albert Einstein Medical Center, Philadelphia, Pa. This paper was presented before the American Society for the Study of Sterility, 18th Annual Meeting, Chicago, Ill., Mar. 30--Apr. 1, 1962.
231
232
PERLOFF
& STEINBERGER
FERTILITY
& STERILITY
studied and photographed with the Zeiss "Photoscope," employing phasecontrast optics. RESULTS
In most preparations one could visualize phase lines between cervical mucus and seminal fluid immediately following approximation of the two fluids (Fig. 1). Shortly thereafter, spermatozoa-filled phalanges could be observed. Phalanges, which uniformly projected into cervical mucus and never in reverse direction, soon developed marked degrees of arborization (Fig. 2), the terminal aspects of which consisted of canals through which only single spermatozoa or at most 2 spermatozoa abreast were able to pass (Fig. 3). Canals appeared to be long finger-like projections of seminal fluid into cervical mucus as evidenced by the ability of spermatozoa which had broken through into the mucus from other areas to pass immediately over andjor under the canals. As a single spermatozoon progressed along a canal, it was soon followed by a column of other spermatozoa. When the leader spermatozoon was blocked in its advance, those which followed ceased their forward motion. With resumption of forward progress by the leader spermatozoon, followers would likewise advance. Spermatozoa were observed to penetrate the substance of cervical mucus, singly or in groups, from the ends or sides of canals as well as from all phase lines between the two fluids. When one spermatozoon traversed a phase line, others in close proximity slipped through the same "opening." It was noted frequently that spermatozoa progressing actively within the substance of cervical mucus stopped abruptly as if they had encountered an impediment which prevented forward progress. Single or multiple spermatozoa so affected increased their activitj; thrashing about as though trying to break through this obstacle. After variable periods of time, either singly or together, they overcame the barrier and were able to progress further into the substance of the mucus. Careful observation of these areas by phase microscopy often revealed what appeared to be intramucus phase lines (Fig. 4). Having confirmed Miller and Kurzrok's thesis of phalangeal formation, we attempted to ascertain whether this was a function of spermatozoal activity, as they postulated, or an inherent characteristic of the two fluids predicated on their physicochemical properties. Accordingly, we introduced azoospermic specimens into the test system and noted development o.f phases and phalanges despite absence of spermatozoa in seminal fluid. These formations were also seen when azoospermic seminal fluid was tested against ovulatory cervical mucus containing large numbers of motile spermatozoa from insemination performed 24 hr. previously (Fig. 5). In such instances spermatozoa were seen to pass freely through phase lines from cervical mucus into azoospermic seminal fluid (Fig. 6). To evaluate further
VoL. 14, No. 2, 1963
SPERM AND CERVICAL
Mucus
233
I
Fig. 1. Phase-line formation at contact zone between seminal fluid and cervical mucus. A, Seminal fluid (streaked appearance of spermatozoa photographed in rapid motion). B, Cervical mucus, arrow pointing to phase line with early phalanx formation. Fig. 2. Arborization of phalanges in cervical mucus. A, Seminal fluid. B, Cervical mucus, arrow pointing to arborization of phalanges. Fig. 3; A, Seminal fluid. B, Cervical mucus, arrow pointing to a "canal" containing spermatozoa. Fig. 4, Cervical mucus 24 hr. after insemination showing intramucus phase lines.
234
PERLOFF
& STEINBERGER
FERTILITY
& STERILITY
Fig. 6. Cervical mucus 24 hr. after insemination forming phase lines and phalanges with azoospermic seminal fluid. A, Seminal fluid. B, Cervical mucus, arrow pointing to early phalanx formation. Fig. 6. Penetration of spermatozoa from cervix mucus into seminal fluid. A, Seminal fluid. B, Cervical mucus, arrow c pointing to sperm moving across phase line from cervical mucus into seminal fluid, arrow d pointing toward phase line. Fig. 7. Phalanx formation between cervical mucus and azoospermic seminal fluid containing India ink. A, Seminal fluid. B, Cervical mucus, arrow pointing to phase line. Fig. 8. Phalanx formation between azoospermic India ink containing seminal fluid and cervical mucus. A, Seminal fluid. B, Cervical mucus, arrow pointing toward India-ink particles in
phalanx.
VoL. 14, No. 2, 1963
SPERM AND CERVICAL
Mucus
235
the role of spermatozoa in phalanges and canal formation, the following experiment was conducted. Azoospermic seminal fluid was mixed with a small amount of India ink, and phase formation with ovulatory mucus was observed (Fig. 7). Phalanges and canals developed readily; India-ink particles penetrated into the canals (Fig. 8) and were also able to traverse phase lines into the cervical mucus. COMMENT
In this communication we have presented observations which confirm Miller and Kurzrok' s concept of microscopically demonstrable phase and phalanx formation at contact zones between ovulatory cervical mucus and seminal fluid. Whereas these authors ascribed formation of phalanges primarily to directed spermatozoal activity, we have provided evidence that phalanges develop not only at the boundary between cervical mucus and normal seminal fluid, but also between cervical mucus and azoospermic seminal fluid, indicating that participation of spermatozoa is not essential. Miller and Kurzrok reported spermatozoal passage from phalanges into cervical mucus. We have seen spermatozoa pass readily into cervical mucus from seminal fluid at any point of contact regardless of presence of phalanges. Consequently, phalangeal formation does not seem to be essential for spermatozoal penetration of cervical mucus. Inert (India-ink) particles suspended in azoospermic seminal fluid pass freely into phalanges and probably are transferred passively across all contact zones between seminal fluid and cervical mucus. Motility, therefore, does not seem to be an absolute prerequisite for penetration of cervical mucus, nor for formation of phalanges. Miller and Kurzrok and others3 • 4 have postulated a "chemotactic" attraction of cervical mucus for spermatozoa. Employing a system composed of spermatozoa containing cervical mucus from insemination 24 hr. previously, and azoospermic seminal fluid, we observed free passage of spermatozoa across contact zones from cervical mucus into seminal fluid. In these preparations, phalanges which formed at contact zones between the two fluids were directed from seminal fluid into cervical mucus, despite the fact that spermatozoa were traveling in reverse direction, lending further support to the suggestion that phalanges are not formed by spermatozoal activity. A chemotactic attraction by cervical mucus for spermatozoa is difficult to envision in view of these observations. In certain instances correlation between in-vitro and in-vivo penetration of cervical mucus by spermatozoa was not good, a phenomenon which requires explanation and is being investigated. Immediately following sexual intercourse, most of the ejaculate flows out
236
PERLOFF & STEINBERGER
FERTILITY & STERILITY
of the vagina, ·and .only a relatively small amount remains in contact with cervical mucus. In view of the relative slowness of cervical-mucus penetration py spermatozoa and rapidity with which spermatozoa are collected in the phalanges and canals, and in view of the tremendous increase in contact surface area occasioned by these formations, one cannot help but speculate as to the following sequence of events: 1. Approximation of ejaculate to cervical mucus 2. Formation of phalanges and canals with consequent trapping of a large number of spermatozoa 3. Gradual penetration of spermatozoa into cervical mucus from these repositories. CONCLUSIONS
When ovulatory cervical mucus and seminal fluid are placed in apposition in vitro, phase lines are formed from which phalanges and canals soon project into cervical mucus. This phenomenon does not depend upon the presence of spermatozoa. Many spermatozoa are trapped quickly in these repositories, from which they gradually penetrate cervical mucus. It is likely that phalanges and canals, in addition to pedorming a storage function, increase the sudace area between the two fluids which facilitate spermatozoal penetration of cervical mucus. Albert Einstein Medical Center York and Tabor Rds. Philadelphia 41, Pa.
REFERENCES I. KmsTELLER, S. Beitrage zu den Bedingungen der Conception. Berl. Klin. W chnschr. 8:315, 1871. 2. MILLER, E. G., JR., and KURZROK, R. Biochemical studies of human semen. III. Factors affecting migration of sperm through the cervix. Am. ]. Obst. & Gynec. 24:19, 1932. 3. SEELIGMANN, L. Weitere Mitteilungen vur Behandlung der Sterilitas Matrimonii. Zentralbl. f. Gyniik. 20:429, 1896. 4. STRASSMANN, P. "Vordringen der Spermien," in Winkel's Handbuch der Geburtschilfe, vol. 1, Part 1, Wiesbaden, 1903, p. 154.