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The interaction of human spermatozoa with cervical mucus in vivo
The interaction mucus in vivo FREDERICK JAMES Davis,
W.
W.
of human spermatozoa with cervical
HANSON,
M.D.
OVERSTREET,
M.D.,
PH.D.*
California
The interaction between human spermatozoa and cervical mucus was studied during 14 cycles of artificial insemination (Al) with a cervical cup. The concentration of spermatozoa in the inseminate was determined as were the percentage of motility, mean swimming speed, and sperm morphology. The percentage of motility, swimming speed, and morphology of spermatozoa in the mucus were determined at 1 hour and 48 hours after Al. The percentage of motile sperm was always higher in the cervical mucus than in the semen. In some cases, spermatozoa appeared to swim faster in the mucus than in the semen but in other cases the reverse was true. In all 14 cases some spermatozoa could be found in the mucus at 48 hours after Al. In general, the percentage of motility and swimming speeds of the cervical sperm remained unchanged over the 4Ehour study interval. The percentage of spematozoa with normal morphology was higher in the cervical mucus than in the semen. This alteration in the composition of the sperm copulation appeared to result from exclusion by the mucus of most classes of abnormal sperm. The morphology of the cervical sperm population was similar at 1 hour and at 48 hours after Al. (AM. J. OBSTET. GYNECOL. 140:173, 1981.)
CERVICAL MUCUS plays a unique role in the physiology of human conception. It protects the portal of entry to the female reproductive tract, blocking the passage of spermatozoa during most of the menstrual cycle. For a brief interval in the preovulatory and periovulatory periods, it facilitates the passage of sperm cells from the vagina to the uterine cavity, and it appears to exercise some selection as to the quality of spermatozoa which are admitted.’ During sperm transport, the endocervix may be a major site of sperm storage in women, and thus it may be the most favorable environment within the entire reproductive tract for prolonged sperm survival.2 The cervical mucus may provide the milieu for the endogenous physiologic events of sperm capacitation, and its unique properties may even induce these changes in fertile sperm cells.” THE
Both the semen and the cervical mucus are easily obtained for clinical assessment. The Sims-Huhner test is a routine procedure in the evaluation of female infertility and provides valuable information on the interaction of spermatozoa and cervical mucus in vivo.-l A therapeutic artificial insemination (AI) program provides the setting in which the times of insemination and observation can be controlled, the properties of the inseminate and cervical mucus can be recorded, and the conditions of exposure between them can be standardized. We have recently begun to study human spermcervical mucus interaction under these conditions. Our initial observations of the effect of cervical mucus on sperm motility and morphology are the subject of this communication.
Material and methods From the Department of Obstetrics and Gynecology and Human Anatomy, School of Medicine, University of California, Davis. Presented by invitation at the Forty-seventh Annual Meeting of the Pacific Coast Obstetrical and Gynecological Society, Monterey, California, October 6-1 I, 1980. Reprint requests: Frederick W. Hanson, M.D., Department of Obstetrics and Gynecology, School of Medicine, University of California, Davis, California 95616. *Recipient HDO0224. 0002-93’78/81/100173+06$00.60/0
of Research
Career
0
Development
1981
The
Award
C. V. Mosby
Co.
Sperm-cervical mucus interaction was studied in 14 cycles of AI. In nine cycles the husband’s semen was used for insemination and in five cycles donor semen was used. Semen was obtained by masturbation and was inseminated within 1 hour of collection. Inseminations were scheduled for the periovulatory period of the menstrual cycle on the basis of menstrual history and previous basal body temperature (BBT) graphs. AI was performed within a 2-day interval before the expected nadir of the BBT. Immediately before AI, 173
174
Hanson
and Overstreet
Fig. 1. Examples of the morphological classes of spermatozoa in this study: (1) normal oval, (2) large. (3) small, (4) tapering, (5) headless, (6) amorphous-head, (7) amorphous-midpiece, (8) amorphoustail, and (9) duplicate.
Table
I. Characteristics
of the inseminates
MotiliQ Husbnnd’.s 1 2 3 4 5
(5%)
.smm:
6 7 8 9 Donor
.wnlLp)?:
10 11 12 13 14
the percentage of motile sperm in the inseminate and their mean swimming speed were determined by timeexposure photomicrography at 37” C.’ The concentration of spermatozoa in the inseminate was determined by hemocytometer counts, and sperm morphology was assessed from Papanicolaou-stained seminal smears. One hundred consecutive cells on each slide were scored for sperm morphology (Fig. 1). All morphologic evaluations were made by a single observer 0. W. 0.) at a magnification of X 1,000 with the aid of an eyepiece micrometer. Spermatozoa with an oval head and a normal midpiece and tail were classified as normal. Abnormal spermatozoa which had a head and tail were classified by head morphology as large, small, tapering, duplicate, or amorphous. Spermatozoa with no head were scored as headless, and those with an abnormal tail
(short, coiled, broken) were scored as amorphous-tail. Spermatozoa with a normal head and tail but with an abnormal midpiece (excess cytoplasm or abnormal headmidpiece angle) were scored as amorphous-midpiece (Fig. 1). Pericervical AI was performed with the use of a cervical cup with stem (cup diameters ranged from Y2 to :36 mm; stem length was 13.5 cm) (Milex, Los Angeles, California). The cervix was visualized with a vaginal speculum, and care was taken to avoid disturbing any mucus protruding from the cervical OS. The speculum was removed and a cup of the appropriate size was placed on the cervix with the stem protruding from the vaginal introitus. The semen was slowly injected from a syringe into the cup via a 12.5 cm catheter thl-cadet1 through the stem. ‘I’he cup was sealed and the patient
Interaction
Sl48
:
No. 1
No. 2
SI
No. 3
of spermatozoa
S
No. 4
with cervical
mucus
175
I 48
No. 5
E 40 30 20 IO 0
S
z 5
I
48
S
148
No. 6
S
No. 7
I
No. 6
s
I
No. 9
S
I 48
No. 10
50-
”
s
I 48 No. 11
S
I 48
No. 12
S
I 48 No. 13
S I 48 No. 14
Fig. 2. Mean swimming speeds (bars) 2 SEM (I) o f sp ermatozoa in semen (S) and in cervical mucus at 1 hour (I) and 48 hours (48) after Al. The number at the foot of each bar refers to the percentage of motile spermatozoa. Values of swimming speed did not differ in the three suspensions, as confirmed by analysis of variance (P < 0.01). Values of the percentage of motility in the mucus were not significantly different at 1 and 48 hours after insemination, and at both times these values were higher than those in the semen (P < O.Ol), as confirmed by analysis of variance after transformation to angles.,
was allowed to leave the examining room and to move about freely during the following hour. One hour after insemination, the patient returned to the examining room and the cervical cup was removed. The exocervix was carefully wiped and approximately 50 ~1 of mucus was collected from the level of the endocervix by means of a polyethylene catheter.” The first 25 ~1 of mucus drawn into the catheter was expressed onto a microscope slide and covered with a No. 1 ti coverglass (22 by 22 mm). After an initial visual observation of the mucus and its contents, the motility of spermatozoa swimming in the mucus was recorded by time-exposure photomicrography.” The coverglass was then used to smear the mucus specimen on the slide, which was subsequently fixed and stained by the Papanicolaou technique for evaluation of sperm morphology. One hundred cells per slide were evaluated by the same procedure used for the seminal smears. The patient returned for a follow-up examination 48 hours after insemination, when the cervical mucus was similarly aspirated and the spermatozoa therein were again evaluated for motility and morphology.
Results The total number of motile spermatozoa in the inseminate ranged from 34 to 410 x 10” (Table I). After
1 hour of semen-mucus contact, there were 20 or more motile sperm per high power microscopic field (X 400) in the mucus in 10 of 14 cycles. Of the four cycles in which fewer sperm were seen at 1 hour, one case seemed attributable to poor semen quality (No. 8), and three cases were associated with thick, cellular mucus (Nos. 6,8, and 14). At 48 hours after insemination, some spermatozoa were present in the mucus in all 14 cases. Sperm numbers in the mucus remained high for 48 hours (2 10 motile sperm per high power field) in six of the 10 cases with initially high sperm concentrations. The percentage of motile sperm in the cervical mucus at 1 hour after insemination was always higher than in the inseminate, and the majority of the cervical sperm remained motile for the ensuing 48 hours (Fig. 2). In some cases the mean swimming speed of the cervical sperm appeared to be higher than that of the seminal sperm (e.g., No. 1). and in one case the sperm were nonprogressive in the mucus when observed initially (No. 13). With this exception, the percentage motility and swimming speeds of the cervical sperm did not appear to change over the 48-hour study interval. A comparison of the morphology of spermatozoa in the inseminate with those recovered in the cervical mucus is given in Fig. 3. The mean percentage of normal oval sperm increased from an average of 50% in
176
Hanson
and Overstreet
phology ensuing
of the cervical 48 hours.
sperm
populaCon
durirlg
the
Comment
Norm01 Ovol ,4 -.-..-~-.___
0
--
-~~~--..
-_._
6 4 2 0
s
I 48
s
Lorge ,4 _~~~~~~
I 48
Small _~-~~ ~--_--~
s
I 48
Headless
s
I 48
Tapering
-
6 -~
s
Ouplicote
I 48
Amorphous Head
s
I
48
Amorphous Midpiece
s
I 48
Amorphous Toil
Fig. 3. Sperm morphology in semen (S) and in cervical mucus at 1 hour (I) and 48 hours (48) after AI. Values are expressed as mean percentages of total sperm after the data from 14 cycles of insemination were pooled. The percentages of normal oval sperm were higher in the mucus at both times than in the semen, as verified by chi-square analysis (P < 0.01). The percentages of large- and small-headed spermatozoa did not differ significantly between semen and mucus. Values of the percentages of spermatozoa in the other morphologic categories were higher in the semen than in the mucus (P < 0.05) but did not differ significantly within the mucus at the two times.
the inseminate to more than 80% in the cervical mucus. This increase in the proportion of normal sperm appeared to be accomplished by the exclusion of sperm from most of the abnormal classes (Fig. 3). There was no strong evidence for a reduction in the proportion of sperm with large or small heads, but the number of such cells studied was small. The alteration in the proportion of normal and abnormal sperm was apparent in the mucus examined initially at 1 hour after insemination and there was no apparent change in the mor-
AI is often pertin-med two or three times during the midcycle, the inseminations usually being spaced approximately 48 hours apart. The rationale f’or this timing is based on two assumptions: first. that the fertile life of’ the human spermatozoon is 4~ I~om-s or longer, and second, that spermatozoa are retail& within the female tract f’or at least a similar length 01‘ time. There is clinical evidence which suggests that human sperm may remain fertile in the woman for several days, although conclusive scientific proof is lacking.’ ‘l‘he ability of’ human sperm to enter and survive in the cervical environment can and should be routinelv evaluated for every patient receiving AI. Our observations indicate that human sperm motility remains relatively rmchanged in the cervical mucus over a #-hour inlerval during midcycle. Therefore, if sperm are able to enter the mucus initially, some will usually remain .1X hours later and can be evaluated by sampling the mucus prior to the next insemination. We now pertbrm such observations routinely, and shorter-term examinations have proved necessary in only a few cases. It should be emphasized that the cervical cup technique is a very ettlcient system for delivery of- spermatozoa to the cervix. l‘he number ot’spwm which remain in the cervical mucus for 48 hours appears to be greater with this technique than with either coitus or intravaginal AI.n During sperm transport in the woman, a great reduction in sperm numbers is accomplished, and most morphologically abnormal cells do not reach the site of f’ertilization in the fallopian tube.g Experiments in vitro have suggested that this may be due to the interaction between spermatozoa and the cervical mucus.’ Observations of the morphology of spermatozoa recovered fi-om the mucus after coitus have provided support for this view.“’ Our observations are the first which directly compare the morphology of spermatozoa in the inseminate with that of the cervical sperm. They confirm the notion that the cervical mucus acts as a biological “filter.” excluding spermatozoa with abnormal morphology. However, our findings differ from previous information on the types of abnormal sperm which are excluded by the mucus. ‘7 I” Mie found no evidence of selection against large or small sperm heads, provided the sperm shape was round or oval. The numbers of spermatozoa in most of the other classes of abnormal morphology were significantly reduced by the mucus. This was true even for “minor” abnormalities such as cytoplasm on the sperm midpiece. The mechanism of sperm exclusion by the cervical
Vdume 140 Number 2
Interaction of spermatozoa with cervical mucus
mucus is unclear. Physical filtration by the mucus microstructure seems unlikely to be the only factor, because of the diversity of head shapes which are excluded. Neither is sperm flagellar activity likely to be a single discriminating characreristic, since many of the types of cells which are excluded may have movement
177
characteristics which are indistinguishable from those of the normal population (unpublished observations). Further experiments will be necessary to examine other biological factors which may mediate sperm entry or exclusion by the mucus, such as the surface properties of the sperm cells.
REFERENCES
1. Perry, G., Glezerman.
M., and Insler, V.: Selective filtration of abnormal spermatozoa by the cervical mucus in vitro, in Insler, V., and Bettendorf, G., editors: The Uterine Cervix in Reproduction, Stuttgart, 1977, George Thiem Verlag, p. 118. 2. Overstreet, J. W., and Katz, D. F.: Sperm transport and capacitation, in Speroff, L., and Simpson, J. L., editors: Gynecology and Obstetrics, New York, vol. 5, Harper & Row, Publishers, Inc. In press. 3. Overstreet, J. W., Gould, J. E., Katz, D. F., and Hanson, F. W.: In vitro capacitation of human spermatozoa after passage through a column of cervical mucus, Fertil. Steril. 34~604, 1980. 4. Moghissi, K. S.: Postcoital test: Physiologic basis, technique, and interpretation, Fertil. Steril. 27: 117, 1976. 5. Overstreet, J. W., Katz, D. F., Hanson, F. W., and Fonseca, 1. R.: A simple inexpensive method for objective assessment of human spe& movement characte>istics, Fertil. Steril. 31: 162. 1979.
Discussion DR. RONALD M. NELSON, Los Angeles, California. With this nice study, the authors have redrawn our attention to the biological function of the cervix in reproduction. It is well established that the cervix plays a major anatomic and physiologic role in the success of sperm migration through the reproductive tract. By the secretion of preovulatory mucus, a favorable environment is created to provide for maximum sperm receptivity, viability, and storage for continuous uppertract insemination. An evaluation of cervical mucus and sperm interaction is an essential part of an infertility examination. In recent years, numerous in vitro studies have greatly improved our knowledge of the physical and biochemical factors that control sperm and mucus interaction. Aithough in vitro penetration tests are advantageous as they can be well controlled, are easily reproduced and objective, artifacts may be easily introduced by laboratory manipulation and the true physiology of insemination may not be accurately represented.
An in vivo postcoital examination is simple to perform and can reveal important information regarding cervical mucus insemination. However, a precise correlation of the postcoital test with semen quality has been limited by the practical inability to determine the quality of the inseminate. In
this
study,
the
problem by performing ity. They found that
authors
have
circumvented
AI with semen of known the
percentage
of
motility
this
qualand
6. Katz, D. F., Overstreet, J. W., and Hanson, F. W.: A new quantitative test for. sperm penetration into cervical mucus, Fertil. Steril. 3% 179, 1980. 7. Austin, C. R.: Sperm fertility, viability and persistence in the female tract, J. Reprod. Fertil. 22:75, 1975. 8. Tredway, D. P., Settlage, D. S. F., Nakamura, R. M., Motoshima, M., Umezaki, C. V., and Mishell, D. R.: Significance of timing for the postcoital evaluation of cervical mucus, AM.J. OBSTET. GYNECOL. 141:387, 1975. 9. Ahlgren, M., Bostrom, K., and Malmqvist, R.: Sperm transport and survival in women with special reference to the fallooian tube. in Hafez. E. S. E.. and Thibault. C. G.. editors: The Biology of Spermatozoa, Basal, 1975, S. Karger AG, p. 63. 10. Fredricsson, B., and Bjork, G.: Morphology of postcoital spermatozoa in the cervical secretion and its clinical significance, Fertil. Steril. 28:841, 1977. ,
1
normal morphology of the sperm was always higher in the cervical mucus than in the semen, and the swimming speed and the percentage of motility of cervical mucus sperm were unchanged up to 48 hours after insemination. Their detailed comparison of the morphology of sperm in the cervical mucus to the semen is an important contribution to our knowledge of spermcervical mucus interaction. My questions for Dr. Hanson involve primarily the design of the study. What were the therapeutic indications for Al by husband in view of the normal semen analysis and good cervical mucus? As a matter of convenience for the patient and physician, a postcoital test is frequently performed at 8 to 12 hours following sexual intercourse. Was that time interval considered in the design of the study? The artificial insemination was performed with a cervical cup applied to the cervix for 1 hour. This technique, although highly efficient, is a significant departure from the physiology of normal intravaginal sperm deposition. The much longer exposure of motile sperm to cervical mucus may explain what would appear
to be the
unusually
high
number
of sperm
seen
in
the cervical mucus after 48 hours. Does the author feel that by protecting the sperm from exposure to a hostile vaginal environment, they may have significantly influenced the results of their study? Has a similar study with the use of an intravaginal insemination technique for comparison been considered? Finally, were sexual intercourse
the
patients 48 hours
instructed to abstain from prior to the insemination
178 Hanson and Overstreet
and for the .4X ltours of the study? Coitus by the husband prior to or during the study could significantl\, alter the findings, particularly in the 3%hour fhllow-up period. Even in the donor-inseminated group, sexual abstinence tnay be important. Quinlivan and Sullivan’ reported that in some cases of‘donor insemination, the husband’s semen tnay contain agglutinating or itntnobili/ing antibodies that adversely affect normal donor spermatozoa. Was the cervical mucus examined fill- rhe prt5etice of’ spermatozoa prior to perfi)rtning insemination in 1x)th study groups? REFERENCE
I. Quinlivan, W. I.., and Sullivan, H.: Spermatozoa] antibodies in human seminal plasma as a cause of failed artificial donor insemination, Fertil. Steril. 28: 1082, 1977. DR. ERLE HENRIKSEN, Los Angeles, Califhrnia. I feel, as do most investigators in the field of’ reproduction, that the essayist has overlooked a very important factor and that is the overwhelming inborn propagative instinct of. the sperm. Tltere are \‘ery few reports describing the problems confronting the sperm, except when it is deposited within the vagina. 1n 19-l 1, I presented before this group, a report, well documented but never published, dealing with impregnation in the absence of admitted exposure to the male. The “Eastern Star” test was employed to determine the authenticity of each case. All cases f’ailed the test. 1 would like to mention three interesting cases. In Case I, described in a Southern medical journal, a young Southern lady was wounded during the battle of Shiloh. ,4 “mini-ball” passed through the testicle of a Northern of’ficer, traversed a meadow, penetrated through 1.5 petticoats, and entered the lady’s abdomen. Nine months later she gave birth to a healthy male child. Although not authenticated, I have always understood that the “mini-ball” was l’ound in the child’s testicle. When critical11 studied, it is amazing that the sperm was still potent, f’ollowing the trials and tribulations involved. .I‘lte second case, reported by the late Harve) Gushing, describes a case occurring during World War 1. Onr of’ his surgical nurses was wounded with a fragtnent of shrapnel. She produced a normal child. One of my reported cases involves a young, single lass, approxitnatel! 11 monrtts pregnant. She vehemently denied exposure. As usual, with such cases. I got up, opened the slttttters. attd studied the Eastern skies. She said. “Doctor, what are you looking for?’ I replied that “the last time this happened, a large and very bright stat
appeared in the East: however, I do not see it IIO~V.” She then adtnitted that “he did put his hand on m\ knee.” Here again, the problems encotttttered ln the sperm, as it moved f’rotn the knee into the vagina and eventually impregnated an ovum. at-e almost unbclievable. She subsequetttl), had a normal child. DR. EMMET LAMB, Stanford, Califorttia. 1s tltc tttetaphor of’ filtration a good one? It the nt~~~t~s 1iltt.rs. thett there should be an increased nuntb~t~ of abnormal sperm in the cervical mucus because the normal forms would have traversed through the cervical mucus. the filter paper in the analogy, and would be in the upper reproductive tract, the filtrate. The authors hale reported a decreased number of’ abnormal f’ornts in the mucus, but they did nor study the fluids in the endotnctrial cavity or in the tube. Studies of the numbers of sperm in the upper reproductive tract ha\Te been ~OIIC in laboratory animals, but I am not aware of any studies in which the morphology of’ the sperttt was examined. A study of that sort would be cottsiderably nto~-c’ difhcult in hutnans, bur necessar) in order to reach the conclusion that the tnucus filters. DR. HANSON (Closing). In answer to Dr. Nelson, tltc indications f’or therapeutic insemination with the 11\1sband’s semen were an attempt to impro\,c the ttutnbcrs of spermato/.oa itt the muct~s and patient desire. It is our opinion that cervical cupping is an el’ficiettt deli\,er\ system of spermatozoa to the ccr\ix. The tinte selected for the postcoital test was based pritnaril) on patient and investigator convenience. M’e detinitel?- feel that the cupping technique protects the sl~ertna~o~oa f’rom the hostile vaginal environment. thereby increasing the numbers of nortnal spermato/o;t which potentiallv could penetrate the cervical mu< IIS. Our patients are instructed to abstain from intercourse for 3 days in advance of’ insemination. during insemination, and for “1 hottrs after insemination. We alwa\s esantined the cervical mucus prior to each insetninat’ion for the presence of spermato/.oa. In attswer to Dr. L,atnb, 1 would ha\c to agree that the use of’the word ftlter in relation to c-crvical IIIIICUS is perhaps ttot correct. As discussed, the mechanism of exclusion of abnortnal spermato/.oa from the mucus is not clear. It may be partly a failure to penetrate the mucus, as well as a washout phenomenon. We do stuclv the IIIUCIIS at multiple levels in the ettdocervical canal and usually see significant differences in sperm numbers, particularly at I hour when tnuctts f’rom higher in the endocervical canal is cotnpared with that from the exocervix.
W.
W.
of human spermatozoa with cervical
HANSON,
M.D.
OVERSTREET,
M.D.,
PH.D.*
California
The interaction between human spermatozoa and cervical mucus was studied during 14 cycles of artificial insemination (Al) with a cervical cup. The concentration of spermatozoa in the inseminate was determined as were the percentage of motility, mean swimming speed, and sperm morphology. The percentage of motility, swimming speed, and morphology of spermatozoa in the mucus were determined at 1 hour and 48 hours after Al. The percentage of motile sperm was always higher in the cervical mucus than in the semen. In some cases, spermatozoa appeared to swim faster in the mucus than in the semen but in other cases the reverse was true. In all 14 cases some spermatozoa could be found in the mucus at 48 hours after Al. In general, the percentage of motility and swimming speeds of the cervical sperm remained unchanged over the 4Ehour study interval. The percentage of spematozoa with normal morphology was higher in the cervical mucus than in the semen. This alteration in the composition of the sperm copulation appeared to result from exclusion by the mucus of most classes of abnormal sperm. The morphology of the cervical sperm population was similar at 1 hour and at 48 hours after Al. (AM. J. OBSTET. GYNECOL. 140:173, 1981.)
CERVICAL MUCUS plays a unique role in the physiology of human conception. It protects the portal of entry to the female reproductive tract, blocking the passage of spermatozoa during most of the menstrual cycle. For a brief interval in the preovulatory and periovulatory periods, it facilitates the passage of sperm cells from the vagina to the uterine cavity, and it appears to exercise some selection as to the quality of spermatozoa which are admitted.’ During sperm transport, the endocervix may be a major site of sperm storage in women, and thus it may be the most favorable environment within the entire reproductive tract for prolonged sperm survival.2 The cervical mucus may provide the milieu for the endogenous physiologic events of sperm capacitation, and its unique properties may even induce these changes in fertile sperm cells.” THE
Both the semen and the cervical mucus are easily obtained for clinical assessment. The Sims-Huhner test is a routine procedure in the evaluation of female infertility and provides valuable information on the interaction of spermatozoa and cervical mucus in vivo.-l A therapeutic artificial insemination (AI) program provides the setting in which the times of insemination and observation can be controlled, the properties of the inseminate and cervical mucus can be recorded, and the conditions of exposure between them can be standardized. We have recently begun to study human spermcervical mucus interaction under these conditions. Our initial observations of the effect of cervical mucus on sperm motility and morphology are the subject of this communication.
Material and methods From the Department of Obstetrics and Gynecology and Human Anatomy, School of Medicine, University of California, Davis. Presented by invitation at the Forty-seventh Annual Meeting of the Pacific Coast Obstetrical and Gynecological Society, Monterey, California, October 6-1 I, 1980. Reprint requests: Frederick W. Hanson, M.D., Department of Obstetrics and Gynecology, School of Medicine, University of California, Davis, California 95616. *Recipient HDO0224. 0002-93’78/81/100173+06$00.60/0
of Research
Career
0
Development
1981
The
Award
C. V. Mosby
Co.
Sperm-cervical mucus interaction was studied in 14 cycles of AI. In nine cycles the husband’s semen was used for insemination and in five cycles donor semen was used. Semen was obtained by masturbation and was inseminated within 1 hour of collection. Inseminations were scheduled for the periovulatory period of the menstrual cycle on the basis of menstrual history and previous basal body temperature (BBT) graphs. AI was performed within a 2-day interval before the expected nadir of the BBT. Immediately before AI, 173
174
Hanson
and Overstreet
Fig. 1. Examples of the morphological classes of spermatozoa in this study: (1) normal oval, (2) large. (3) small, (4) tapering, (5) headless, (6) amorphous-head, (7) amorphous-midpiece, (8) amorphoustail, and (9) duplicate.
Table
I. Characteristics
of the inseminates
MotiliQ Husbnnd’.s 1 2 3 4 5
(5%)
.smm:
6 7 8 9 Donor
.wnlLp)?:
10 11 12 13 14
the percentage of motile sperm in the inseminate and their mean swimming speed were determined by timeexposure photomicrography at 37” C.’ The concentration of spermatozoa in the inseminate was determined by hemocytometer counts, and sperm morphology was assessed from Papanicolaou-stained seminal smears. One hundred consecutive cells on each slide were scored for sperm morphology (Fig. 1). All morphologic evaluations were made by a single observer 0. W. 0.) at a magnification of X 1,000 with the aid of an eyepiece micrometer. Spermatozoa with an oval head and a normal midpiece and tail were classified as normal. Abnormal spermatozoa which had a head and tail were classified by head morphology as large, small, tapering, duplicate, or amorphous. Spermatozoa with no head were scored as headless, and those with an abnormal tail
(short, coiled, broken) were scored as amorphous-tail. Spermatozoa with a normal head and tail but with an abnormal midpiece (excess cytoplasm or abnormal headmidpiece angle) were scored as amorphous-midpiece (Fig. 1). Pericervical AI was performed with the use of a cervical cup with stem (cup diameters ranged from Y2 to :36 mm; stem length was 13.5 cm) (Milex, Los Angeles, California). The cervix was visualized with a vaginal speculum, and care was taken to avoid disturbing any mucus protruding from the cervical OS. The speculum was removed and a cup of the appropriate size was placed on the cervix with the stem protruding from the vaginal introitus. The semen was slowly injected from a syringe into the cup via a 12.5 cm catheter thl-cadet1 through the stem. ‘I’he cup was sealed and the patient
Interaction
Sl48
:
No. 1
No. 2
SI
No. 3
of spermatozoa
S
No. 4
with cervical
mucus
175
I 48
No. 5
E 40 30 20 IO 0
S
z 5
I
48
S
148
No. 6
S
No. 7
I
No. 6
s
I
No. 9
S
I 48
No. 10
50-
”
s
I 48 No. 11
S
I 48
No. 12
S
I 48 No. 13
S I 48 No. 14
Fig. 2. Mean swimming speeds (bars) 2 SEM (I) o f sp ermatozoa in semen (S) and in cervical mucus at 1 hour (I) and 48 hours (48) after Al. The number at the foot of each bar refers to the percentage of motile spermatozoa. Values of swimming speed did not differ in the three suspensions, as confirmed by analysis of variance (P < 0.01). Values of the percentage of motility in the mucus were not significantly different at 1 and 48 hours after insemination, and at both times these values were higher than those in the semen (P < O.Ol), as confirmed by analysis of variance after transformation to angles.,
was allowed to leave the examining room and to move about freely during the following hour. One hour after insemination, the patient returned to the examining room and the cervical cup was removed. The exocervix was carefully wiped and approximately 50 ~1 of mucus was collected from the level of the endocervix by means of a polyethylene catheter.” The first 25 ~1 of mucus drawn into the catheter was expressed onto a microscope slide and covered with a No. 1 ti coverglass (22 by 22 mm). After an initial visual observation of the mucus and its contents, the motility of spermatozoa swimming in the mucus was recorded by time-exposure photomicrography.” The coverglass was then used to smear the mucus specimen on the slide, which was subsequently fixed and stained by the Papanicolaou technique for evaluation of sperm morphology. One hundred cells per slide were evaluated by the same procedure used for the seminal smears. The patient returned for a follow-up examination 48 hours after insemination, when the cervical mucus was similarly aspirated and the spermatozoa therein were again evaluated for motility and morphology.
Results The total number of motile spermatozoa in the inseminate ranged from 34 to 410 x 10” (Table I). After
1 hour of semen-mucus contact, there were 20 or more motile sperm per high power microscopic field (X 400) in the mucus in 10 of 14 cycles. Of the four cycles in which fewer sperm were seen at 1 hour, one case seemed attributable to poor semen quality (No. 8), and three cases were associated with thick, cellular mucus (Nos. 6,8, and 14). At 48 hours after insemination, some spermatozoa were present in the mucus in all 14 cases. Sperm numbers in the mucus remained high for 48 hours (2 10 motile sperm per high power field) in six of the 10 cases with initially high sperm concentrations. The percentage of motile sperm in the cervical mucus at 1 hour after insemination was always higher than in the inseminate, and the majority of the cervical sperm remained motile for the ensuing 48 hours (Fig. 2). In some cases the mean swimming speed of the cervical sperm appeared to be higher than that of the seminal sperm (e.g., No. 1). and in one case the sperm were nonprogressive in the mucus when observed initially (No. 13). With this exception, the percentage motility and swimming speeds of the cervical sperm did not appear to change over the 48-hour study interval. A comparison of the morphology of spermatozoa in the inseminate with those recovered in the cervical mucus is given in Fig. 3. The mean percentage of normal oval sperm increased from an average of 50% in
176
Hanson
and Overstreet
phology ensuing
of the cervical 48 hours.
sperm
populaCon
durirlg
the
Comment
Norm01 Ovol ,4 -.-..-~-.___
0
--
-~~~--..
-_._
6 4 2 0
s
I 48
s
Lorge ,4 _~~~~~~
I 48
Small _~-~~ ~--_--~
s
I 48
Headless
s
I 48
Tapering
-
6 -~
s
Ouplicote
I 48
Amorphous Head
s
I
48
Amorphous Midpiece
s
I 48
Amorphous Toil
Fig. 3. Sperm morphology in semen (S) and in cervical mucus at 1 hour (I) and 48 hours (48) after AI. Values are expressed as mean percentages of total sperm after the data from 14 cycles of insemination were pooled. The percentages of normal oval sperm were higher in the mucus at both times than in the semen, as verified by chi-square analysis (P < 0.01). The percentages of large- and small-headed spermatozoa did not differ significantly between semen and mucus. Values of the percentages of spermatozoa in the other morphologic categories were higher in the semen than in the mucus (P < 0.05) but did not differ significantly within the mucus at the two times.
the inseminate to more than 80% in the cervical mucus. This increase in the proportion of normal sperm appeared to be accomplished by the exclusion of sperm from most of the abnormal classes (Fig. 3). There was no strong evidence for a reduction in the proportion of sperm with large or small heads, but the number of such cells studied was small. The alteration in the proportion of normal and abnormal sperm was apparent in the mucus examined initially at 1 hour after insemination and there was no apparent change in the mor-
AI is often pertin-med two or three times during the midcycle, the inseminations usually being spaced approximately 48 hours apart. The rationale f’or this timing is based on two assumptions: first. that the fertile life of’ the human spermatozoon is 4~ I~om-s or longer, and second, that spermatozoa are retail& within the female tract f’or at least a similar length 01‘ time. There is clinical evidence which suggests that human sperm may remain fertile in the woman for several days, although conclusive scientific proof is lacking.’ ‘l‘he ability of’ human sperm to enter and survive in the cervical environment can and should be routinelv evaluated for every patient receiving AI. Our observations indicate that human sperm motility remains relatively rmchanged in the cervical mucus over a #-hour inlerval during midcycle. Therefore, if sperm are able to enter the mucus initially, some will usually remain .1X hours later and can be evaluated by sampling the mucus prior to the next insemination. We now pertbrm such observations routinely, and shorter-term examinations have proved necessary in only a few cases. It should be emphasized that the cervical cup technique is a very ettlcient system for delivery of- spermatozoa to the cervix. l‘he number ot’spwm which remain in the cervical mucus for 48 hours appears to be greater with this technique than with either coitus or intravaginal AI.n During sperm transport in the woman, a great reduction in sperm numbers is accomplished, and most morphologically abnormal cells do not reach the site of f’ertilization in the fallopian tube.g Experiments in vitro have suggested that this may be due to the interaction between spermatozoa and the cervical mucus.’ Observations of the morphology of spermatozoa recovered fi-om the mucus after coitus have provided support for this view.“’ Our observations are the first which directly compare the morphology of spermatozoa in the inseminate with that of the cervical sperm. They confirm the notion that the cervical mucus acts as a biological “filter.” excluding spermatozoa with abnormal morphology. However, our findings differ from previous information on the types of abnormal sperm which are excluded by the mucus. ‘7 I” Mie found no evidence of selection against large or small sperm heads, provided the sperm shape was round or oval. The numbers of spermatozoa in most of the other classes of abnormal morphology were significantly reduced by the mucus. This was true even for “minor” abnormalities such as cytoplasm on the sperm midpiece. The mechanism of sperm exclusion by the cervical
Vdume 140 Number 2
Interaction of spermatozoa with cervical mucus
mucus is unclear. Physical filtration by the mucus microstructure seems unlikely to be the only factor, because of the diversity of head shapes which are excluded. Neither is sperm flagellar activity likely to be a single discriminating characreristic, since many of the types of cells which are excluded may have movement
177
characteristics which are indistinguishable from those of the normal population (unpublished observations). Further experiments will be necessary to examine other biological factors which may mediate sperm entry or exclusion by the mucus, such as the surface properties of the sperm cells.
REFERENCES
1. Perry, G., Glezerman.
M., and Insler, V.: Selective filtration of abnormal spermatozoa by the cervical mucus in vitro, in Insler, V., and Bettendorf, G., editors: The Uterine Cervix in Reproduction, Stuttgart, 1977, George Thiem Verlag, p. 118. 2. Overstreet, J. W., and Katz, D. F.: Sperm transport and capacitation, in Speroff, L., and Simpson, J. L., editors: Gynecology and Obstetrics, New York, vol. 5, Harper & Row, Publishers, Inc. In press. 3. Overstreet, J. W., Gould, J. E., Katz, D. F., and Hanson, F. W.: In vitro capacitation of human spermatozoa after passage through a column of cervical mucus, Fertil. Steril. 34~604, 1980. 4. Moghissi, K. S.: Postcoital test: Physiologic basis, technique, and interpretation, Fertil. Steril. 27: 117, 1976. 5. Overstreet, J. W., Katz, D. F., Hanson, F. W., and Fonseca, 1. R.: A simple inexpensive method for objective assessment of human spe& movement characte>istics, Fertil. Steril. 31: 162. 1979.
Discussion DR. RONALD M. NELSON, Los Angeles, California. With this nice study, the authors have redrawn our attention to the biological function of the cervix in reproduction. It is well established that the cervix plays a major anatomic and physiologic role in the success of sperm migration through the reproductive tract. By the secretion of preovulatory mucus, a favorable environment is created to provide for maximum sperm receptivity, viability, and storage for continuous uppertract insemination. An evaluation of cervical mucus and sperm interaction is an essential part of an infertility examination. In recent years, numerous in vitro studies have greatly improved our knowledge of the physical and biochemical factors that control sperm and mucus interaction. Aithough in vitro penetration tests are advantageous as they can be well controlled, are easily reproduced and objective, artifacts may be easily introduced by laboratory manipulation and the true physiology of insemination may not be accurately represented.
An in vivo postcoital examination is simple to perform and can reveal important information regarding cervical mucus insemination. However, a precise correlation of the postcoital test with semen quality has been limited by the practical inability to determine the quality of the inseminate. In
this
study,
the
problem by performing ity. They found that
authors
have
circumvented
AI with semen of known the
percentage
of
motility
this
qualand
6. Katz, D. F., Overstreet, J. W., and Hanson, F. W.: A new quantitative test for. sperm penetration into cervical mucus, Fertil. Steril. 3% 179, 1980. 7. Austin, C. R.: Sperm fertility, viability and persistence in the female tract, J. Reprod. Fertil. 22:75, 1975. 8. Tredway, D. P., Settlage, D. S. F., Nakamura, R. M., Motoshima, M., Umezaki, C. V., and Mishell, D. R.: Significance of timing for the postcoital evaluation of cervical mucus, AM.J. OBSTET. GYNECOL. 141:387, 1975. 9. Ahlgren, M., Bostrom, K., and Malmqvist, R.: Sperm transport and survival in women with special reference to the fallooian tube. in Hafez. E. S. E.. and Thibault. C. G.. editors: The Biology of Spermatozoa, Basal, 1975, S. Karger AG, p. 63. 10. Fredricsson, B., and Bjork, G.: Morphology of postcoital spermatozoa in the cervical secretion and its clinical significance, Fertil. Steril. 28:841, 1977. ,
1
normal morphology of the sperm was always higher in the cervical mucus than in the semen, and the swimming speed and the percentage of motility of cervical mucus sperm were unchanged up to 48 hours after insemination. Their detailed comparison of the morphology of sperm in the cervical mucus to the semen is an important contribution to our knowledge of spermcervical mucus interaction. My questions for Dr. Hanson involve primarily the design of the study. What were the therapeutic indications for Al by husband in view of the normal semen analysis and good cervical mucus? As a matter of convenience for the patient and physician, a postcoital test is frequently performed at 8 to 12 hours following sexual intercourse. Was that time interval considered in the design of the study? The artificial insemination was performed with a cervical cup applied to the cervix for 1 hour. This technique, although highly efficient, is a significant departure from the physiology of normal intravaginal sperm deposition. The much longer exposure of motile sperm to cervical mucus may explain what would appear
to be the
unusually
high
number
of sperm
seen
in
the cervical mucus after 48 hours. Does the author feel that by protecting the sperm from exposure to a hostile vaginal environment, they may have significantly influenced the results of their study? Has a similar study with the use of an intravaginal insemination technique for comparison been considered? Finally, were sexual intercourse
the
patients 48 hours
instructed to abstain from prior to the insemination
178 Hanson and Overstreet
and for the .4X ltours of the study? Coitus by the husband prior to or during the study could significantl\, alter the findings, particularly in the 3%hour fhllow-up period. Even in the donor-inseminated group, sexual abstinence tnay be important. Quinlivan and Sullivan’ reported that in some cases of‘donor insemination, the husband’s semen tnay contain agglutinating or itntnobili/ing antibodies that adversely affect normal donor spermatozoa. Was the cervical mucus examined fill- rhe prt5etice of’ spermatozoa prior to perfi)rtning insemination in 1x)th study groups? REFERENCE
I. Quinlivan, W. I.., and Sullivan, H.: Spermatozoa] antibodies in human seminal plasma as a cause of failed artificial donor insemination, Fertil. Steril. 28: 1082, 1977. DR. ERLE HENRIKSEN, Los Angeles, Califhrnia. I feel, as do most investigators in the field of’ reproduction, that the essayist has overlooked a very important factor and that is the overwhelming inborn propagative instinct of. the sperm. Tltere are \‘ery few reports describing the problems confronting the sperm, except when it is deposited within the vagina. 1n 19-l 1, I presented before this group, a report, well documented but never published, dealing with impregnation in the absence of admitted exposure to the male. The “Eastern Star” test was employed to determine the authenticity of each case. All cases f’ailed the test. 1 would like to mention three interesting cases. In Case I, described in a Southern medical journal, a young Southern lady was wounded during the battle of Shiloh. ,4 “mini-ball” passed through the testicle of a Northern of’ficer, traversed a meadow, penetrated through 1.5 petticoats, and entered the lady’s abdomen. Nine months later she gave birth to a healthy male child. Although not authenticated, I have always understood that the “mini-ball” was l’ound in the child’s testicle. When critical11 studied, it is amazing that the sperm was still potent, f’ollowing the trials and tribulations involved. .I‘lte second case, reported by the late Harve) Gushing, describes a case occurring during World War 1. Onr of’ his surgical nurses was wounded with a fragtnent of shrapnel. She produced a normal child. One of my reported cases involves a young, single lass, approxitnatel! 11 monrtts pregnant. She vehemently denied exposure. As usual, with such cases. I got up, opened the slttttters. attd studied the Eastern skies. She said. “Doctor, what are you looking for?’ I replied that “the last time this happened, a large and very bright stat
appeared in the East: however, I do not see it IIO~V.” She then adtnitted that “he did put his hand on m\ knee.” Here again, the problems encotttttered ln the sperm, as it moved f’rotn the knee into the vagina and eventually impregnated an ovum. at-e almost unbclievable. She subsequetttl), had a normal child. DR. EMMET LAMB, Stanford, Califorttia. 1s tltc tttetaphor of’ filtration a good one? It the nt~~~t~s 1iltt.rs. thett there should be an increased nuntb~t~ of abnormal sperm in the cervical mucus because the normal forms would have traversed through the cervical mucus. the filter paper in the analogy, and would be in the upper reproductive tract, the filtrate. The authors hale reported a decreased number of’ abnormal f’ornts in the mucus, but they did nor study the fluids in the endotnctrial cavity or in the tube. Studies of the numbers of sperm in the upper reproductive tract ha\Te been ~OIIC in laboratory animals, but I am not aware of any studies in which the morphology of’ the sperttt was examined. A study of that sort would be cottsiderably nto~-c’ difhcult in hutnans, bur necessar) in order to reach the conclusion that the tnucus filters. DR. HANSON (Closing). In answer to Dr. Nelson, tltc indications f’or therapeutic insemination with the 11\1sband’s semen were an attempt to impro\,c the ttutnbcrs of spermato/.oa itt the muct~s and patient desire. It is our opinion that cervical cupping is an el’ficiettt deli\,er\ system of spermatozoa to the ccr\ix. The tinte selected for the postcoital test was based pritnaril) on patient and investigator convenience. M’e detinitel?- feel that the cupping technique protects the sl~ertna~o~oa f’rom the hostile vaginal environment. thereby increasing the numbers of nortnal spermato/o;t which potentiallv could penetrate the cervical mu< IIS. Our patients are instructed to abstain from intercourse for 3 days in advance of’ insemination. during insemination, and for “1 hottrs after insemination. We alwa\s esantined the cervical mucus prior to each insetninat’ion for the presence of spermato/.oa. In attswer to Dr. L,atnb, 1 would ha\c to agree that the use of’the word ftlter in relation to c-crvical IIIIICUS is perhaps ttot correct. As discussed, the mechanism of exclusion of abnortnal spermato/.oa from the mucus is not clear. It may be partly a failure to penetrate the mucus, as well as a washout phenomenon. We do stuclv the IIIUCIIS at multiple levels in the ettdocervical canal and usually see significant differences in sperm numbers, particularly at I hour when tnuctts f’rom higher in the endocervical canal is cotnpared with that from the exocervix.