Modern Trends POSTCOITAL TEST: PHYSIOLOGIC BASIS, TECHNIQUE, AND INTERPRETATION

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1 FERTILITY AND STERILITY Copyright 1976 The American Fertility Society Modern Trends Vol. 27, No. 2, February 1976 Printed in U.S.A. POSTCOITAL TEST: PHYSIOLOGIC BASIS, TECHNIQUE, AND INTERPRETATION KAMRAN S. MOGHISSI, M.D. Department of Gynecology-Obstetrics, Wayne State University School of Medicine, and C. S. Mott Center for Human Growth and Development, Detroit, Michigan An important step in studying female infertility is the investigation of cervical function. Abnormalities of the cervix and its secretion are reported to be responsible for infertility in approximately 15 to 30% of women, but among properly investigated infertile couples, the true incidence is probably not greater than 5 to 10%. During coitus, 200 to 500 million spermatozoa are deposited on the cervix and posterior vaginal fornix. Human semen coagulates immediately after ejaculation and traps most sperm cells until seminal proteolytic enzymes bring about liquefaction. The first portion of the ejaculate, however, contains the highest concentration of spermatozoa (three-fourths in man) which, under favorable conditions, promptly penetrate cervical mucus. 1 The intermixing of the ejaculate and cervical mucus which is caused by penile movements and displacement of the cervical mucus column may aid this process. The vaginal content is usually acidic, with a ph of approximately 3 to 4. However, the cervical secretion coats the upper part of the vagina and its fornices and increases considerably the alkalinity of the vaginal milieu, providing a favorable medium for spermatozoa and apparently promoting their longevity. Sperm migration through the cervix involves three distinct but interrelated factors: (1) the ability of spermatozoa to penetrate the mucus; (2) the unique structure and composition of cervical Received September 30, mucus which guides, nourishes, and protects the sperm; and (3) the morphologic configuration of cervical crypts which contribute to the storage and preservation of spermatozoa in the cervical canal and their sustained and prolonged release into the upper tract. The human spermatozoon is endowed with intrinsic motility, a property essential for penetrating cervical mucus and subsequent fertilization. There is no evidence that immotile or dead sperm can either pass through the human cervix or effect fertilization. Spermatozoa are highly active cells possessing metabolic, glycolytic, and respiratory enzymes. They are capable of metabolizing a variety of exogenous and some endogenous substrates. Since spermatozoa possess a negligible reserve of endogenous glycogen, they must depend on extracellular carbohydrate for their energy requirements during their stay in or passage through the female reproductive tract. It is not known to what extent sperm survival and motility in cervical mucus are influenced by the amount of utilizable carbohydrates or other sperm nutrients present in cervical secretion. In vitro studies suggest that the viability of sperm is related to the glucose level of cervical mucus. Furthermore, a decrease in the glucose concentration of cervical mucus has been found in many infertile patients. Kellerman and Weid 2 believe that unexplained cervical hostility may reflect a deficient glucose content in cervical mucus.

2 118 MOGHISSI February 1976 Human seminal plasma contains a chymotrypsin-like enzyme and peptidase activity. The sperm acrosome is also coated with a chymotrypsin-like enzyme. In addition, a trypsin-like enzyme (aerosin) and hyaluronidase activity have been demonstrated in the sperm head. 3 4 CERVICAL SECRETION Cervical mucus is a complex secretion produced continously by the secretory cells of the endocervix. A small amount of endometrial, tubal, and, possibly, follicular fluids may also contribute to the cervical mucus pool. In addition, cellular debris from uterine and cervical epithelia and leukocytes are present. Cervical mucus is a heterogeneous secretion which has a number of rheologic properties such as viscosity, flow elasticity, spinnbarkheit, thixotropy, and tack, or stickiness. The most important constituent of cervical mucus is a hydrogel, rich in carbohydrates and consisting of glycoproteins of the mucin type. 5 9 Most of the physical properties of cervical mucus are due to these mucins. Biochemical and biophysical studies have demonstrated that cervical mucus is a fibrillar system consisting of subunits which are made of a peptide core and oligosaccharide side chains. Proteolytic enzymes such as trypsin, chymotrypsin, and pronase have been found to hydrolyze human mucus and mucins, to produce certain physical and chemical changes, and to accelerate sperm migration in vitro. 10 Ovarian hormones regulate the secretion of cervical mucus, estrogen stimulates the production of copious amounts of watery mucus, and progesterone (or progestogen) inhibits the secretory activity of cervical epithelial cells. The physical properties and certain chemical constituents of cervical mucus show cyclic variations. Cyclic alterations in the constituents of cervical mucus may influence sperm penetrability, nutrition, and survival. Figure 1 shows serial determinations of some important properties of human cervical mucus in relation to pituitary and ovarian hormones and sperm penetration in 10 women during normal menstrual cycles. These data clearly demonstrate that optimal changes in cervical mucus properties, such as greatest increases in quantity, spinnbarkheit, ferning, and ph, and decreases in viscosity and cell content, occur immediately prior to ovulation and are reversed after ovulation. Preovulatory mucus is most receptive to sperm penetration. 1 L 12 MacDonald and Lumley 13 have suggested that the proportion of saline in the cervical secretion directly determines the consistency of the mucus and the rate of sperm penetration. Sperm penetrability of human cervical mucus begins approximately on the 9th day of a normal cycle and increases gradually to a peak at ovulation. It is usually inhibited within 1 or 2 days after ovulation, but may persist to a lesser degree for a longer period In some women, sperm penetrability occurs only during a limited period of the menstrual cycle (Fig. 2). Individual variations are common. The human uterine cervix is a thickwalled cylindric structure which tapers off at its inferior extremity. The basic epithelial structure of the cervical mucosa is an intricate system of clefts or grooves which, grouped together, give an illusory impression of glands. These clefts may run in an oblique, transverse, or longitudinal direction, but never cross one another, although they may bifurcate or extend downward Cervical crypts are believed to act as a sperm reservoir. Spermatozoa, guided by the line of strain of cervical mucus, are led to cervical crypts, where they are stored and fed to the upper tract for many hours following coitus.

3 I 'I Vol. 27, No.2 POSTCOITAL TEST 119 Days of Cycle & & _, I& ~~ Sperm "f ~:.~rm~~: ~ '''1 ;iph~ ' :r~~fenq~ ot.. ~... w ~~~ :l(+)~ 0 '.~:; ;:; ~. ±~+~~. 4~~:. 2 i (+)Viscosity : <r ' ' ' ' ' I ' ' ' ' ' ' ' j~, 3AI~' r~m)~ -14 i -!'o i -6 I -~ I +~ I +6 [ +fo f.:. I ~ t6 Days of Cycle oa,o of Cycle z +Z I +12 j 8.8. TEMP i.. ~ i~~~ 'i ~J ~ 1 Estriol E 1 : ~ FIG. 1. Composite profile of sperm penetration, gonadotropin and progesterone, urinary estrogens and pregnanediol, basal body temperature (J3B. TEMP), karyopyknotic index (KPI) of vaginal cytology, and cervical mucus properties throughout the menstrual cycles of 10 normal women. Day 0, day of luteinizing hormone <LH) peak (dotted line). The vertical bars represent 1 SEM. F 1 and F 2 indicate the number of sperm in the first and second microscopic fields (x 200) from interface, 15 minutes after the start of the in vitro sperm-cervical mucus penetration test. FSH, Folliclestimulating hormone. (From Moghissi et al. 12)

4 120 MOGHISSI February 1976 ~ l 20- SPERM - PENETRATION SPINNBARKEIT 14l:: 1 of- (em) 6~ 2f- FERN TEST l I ' ~~ ~F2 L rt +4.,.,. +2-.,...,..,.., \.. 'T,..,.. ph 7~ CELLS it' * 0 t-+ IH I+ ilt * VISCOSITY +~ ~ H It if+ if+ QUANTITY * +tt* DAY OF CYCLE c 1~ p BBT J... ~ r-- ~...-. PERCENT 60f- PROTEINS Q Albumin IIIII IIIII -... t... i'\ ~ l K ~,c: ~ @ Globulin " ',J... Mucoid(N.M.F,) 20- ~,, {... t? I ~ o... "' DAY OF CYCLE FIG. 2. Correlation of basal body temperature!bbt) with cervical mucus properties and sperm penetrability in vitro. F 1 and F 2 indicate the number of spermatozoa in the first and second microscopic fields (X 200) from interface 15 minutes after the start of the in vitro spermcervical mucus penetration test. The arrow shows the probable time of ovulation. N.M.F., Nonmigrating fraction. Note the significant sperm penetrability evident only on the day of ovulation. (From Moghissi. 14 ) b \, '~"., INVESTIGATION OF THE CERVICAL FACTOR IN INFERTILITY Spermatozoa are, at all times, suspended in a fluid medium. The interaction of sperm with the fluids of the female reproductive tract is of critical importance for the survival and functional ability of spermatozoa. Unfortunately, there is no practical method for the evaluation of human uterine and tubal fluids and the study of their effect on sperm. Cervical mucus, however, is readily available for sampling and studies. Evaluation of sperm-cervical mucus interaction, therefore, must be included in an infertility investigation. A stepwise plan for evaluation of the cervical factor in infertility is shown in Figure 3. Semen analysis and examination of cervical mucus should precede postcoital tests and more specific in vitro studies. Many laboratories are unfamiliar with, or are ill-prepared to perform, an adequate semen analysis. Information regarding semen volume and appearance, liquefaction time, sperm density, immediate and delayed motility, quality of motility, eosin stainability, percentage of different morphologic types (seminal cytology), presence of pus cells, agglutination, and other changes must appear on every report.

5 Vol. 27, No.2 POSTCOITAL TEST 121 Evaluation of Semen Analysis / Cervical Mucus""' (C M) ""' Postcoital Test (Fractional) Husband's sperm vs Donor CM Early (2-Bh) Late (1B-24h) I In vitro sperm-cm Penetration Test Slide Test Capillary Tube Test I Cross-match Test / ~ Donor's sperm vs Wife's CM FIG. 3. Steps in evaluation of the cervical factor in infertility. Evaluation of Cervical Mucus The condition of the cervical mucus greatly influences sperm receptivity; therefore, it should be evaluated accurately before a postcoital test is performed. Preovulatory mucus receptive to sperm penetration is profuse, thin, clear, acellular, and alkaline. It exhibits a 4+ ferning (crystallization) and high spinnbarkheit. Several techniques for the collection of cervical mucus have been described. The methods most commonly used are aspiration with a tuberculin syringe (without needle), pipette, or polyethylene tube or sampling with a mucus forceps. Clinical examination of cervical mucus includes determination of amount, viscosity, cellularity, ph, ferning, and spinnbarkheit, and culture studies if infection is suspected. Ferning is demonstrated by spreading the cervical mucus on a glass slide and allowing it to dry. It is customarily graded 0 to 4+, depending on the extent of crystal formation. Spinnbarkheit is measured by placing an adequate amount of cervical mucus on a microscope slide, covering it with a cover slip, and drawing the mucus between them. An estimate of spinnbarkheit (in centimeters) is made by measuring the length of the thread before it breaks. Postcoital Test (Sims-Huhner Test) "If we take a drop of semen from the vagina immediately after sexual intercourse and place it under the microscope, we shall see the hurried movements of seemingly thousands of spermatozoa. But this is not the best way of studying the phenomena of their movements. The best plan is to take a drop of mucus from the canal of a perfectly normal cervix uteri some fifteen or twenty hours after sexual intercourse. We shall then be better able to examine the spermatozoa; for we shall see them in the fluid that serves as the means of their finding their way towards the ovum. We shall find them moving more slowly, more cautiously, if the term may be allowed." Thus wrote Marion Sims in 1866, when he first described the postcoital (PC) test.18 Sims recognized the importance of sperm motility and timing of the test, performed immediate postcoital tests on many women, and found sperm in the cervical mucus within a few minutes after coitus. He further observed the presence of live spermatozoa in the cervix for 36 to 48 hours after intercourse. To Huhner, however, goes the credit for popularizing the test as an index of cervical mucus-sperm interaction. The Sims Huhner test is now considered an integral part of infertility investigation but, despite its popularity, there is a lack of standardization, and disagreement remains on how to interpret the results. Timing. Postcoital tests should be performed as closely as possible to the time of ovulation, as determined by usual clinical means (basal body temperature, cervical mucus changes, and vaginal cytology). Each couple is instructed to abstain from sexual intercourse for 2 days prior to the test, which is performed

6 122 MOGHISSI February ,..., RiJ endocenix (ijjternales)...,;...,...,;_ 3. LOlli emleeervix h cenix FIG. 4. Technique of the postcoital test using a tuberculin syringe (without needle). Samples are obtained from (1) the vaginal pool, (2) external cervical os, (3) lower part of the endocervical canal, and (4) upper part of the cervical canal (near the internal os). Endometrial aspiration is optional. approximately 6 to 8 hours after intercourse. A satisfactory test may be repeated at a longer interval (18 to 24 hours). The presence of an adequate number of motile spermatozoa at this stage in the endocervix suggests favorable mucus and adequate survival of sperm in the cervix and excludes the cervical factor as a cause of infertility. When the initial test yields poor results, the second test is planned 1 to 3 hours postcoitum. Techniques of the PC test. A nonlubricated speculum is inserted into the vagina and a sample of the posterior vaginal fornix pool is aspirated with a tuberculin syringe (without needle). With a different syringe, samples of cervical mucus are obtained from the exocervix and lower and upper endocervical canal (Fig. 4). These are placed on a separate glass slide, covered with a cover slip, and examined under the microscope at x 200 and x 400. If the exocervical mucus is covered with cells, debris, and vaginal content, the cervix should be wiped dry with cotton prior to obtaining the endocervical specimen. Interpretation. Interpreting the postcoital test requires an understanding of cervical function and sperm transport. The cervix and its secretion act as a biologic valve which allows the entry of sperm into the uterus at midcycle and bars their admission at other times. Cervical mucus protects sperm from the hostile environment ofthe vagina and from being phagocytosed. The mucus also may supplement the energy requirements of sperm, act as a filter to retain abnormal and sluggish sperm, and provide the proper milieu for sperm capacitation. Within seconds, ejaculated sperm enter midcycle cervical mucus. Subsequent migration through the cervical canal is accomplished principally by intrinsic motility. It may be influenced also by the proteolytic activity of seminal plasma and sperm, phalanx formation, and orientation of strands of cervical mucin. The latter phenomenon may be responsible for the storage of sperm in the cervical crypts and their gradual release over an extended period into the uterus and oviducts. The function of the cervix as a sperm reservoir is therefore

7 Vol. 27, No.2 POSTCOITAL TEST 123 of considerable importance in fertility. Only rarely does coitus occur at the time of ovulation. In most instances, the union of gametes depends on a constant supply of sperm at the site offertilization for some hours before and after ovulation. Following coitus, a gradient is established within the cervix which is entirely time-dependent. With increasing intervals between coitus and examination, there is an orderly progression of the sperm population from the lower to the upper part of the canal. On the basis of the cervical mucus examination following artificial insemination, Tredway et al. 19 suggested that the most appropriate time to perform a postcoital test is 2.5 hours after intercourse, since they found the largest sperm population in the mucus at this time. The purpose of a postcoital test is not only the determination of a sufficient number of active spermatozoa in the cervical mucus but also to evaluate sperm survival and behavior many hours after coitus (reservoir role). Therefore, 6 to 8 hours PC represent a balanced time for determining both sperm density and longevity. Earlier timing may be reserved for those subjects who have negative or abnormal tests. With these facts in mind, postcoital tests may be interpreted on a rational basis. Vaginal Pool Sample. Spermatozoa are usually destroyed in the vagina within 12 hours. 20 Therefore, the purpose of examining the vaginal pool sample is to ensure that semen has actually been deposited in the vagina. Exocervical and Low Cervical Samples. The number of sperm in the lower part of the cervical canal varies with the time elapsed after coitus. Within 2 to 3 hours after intercourse there is a large accumulation of sperm in the lower part of the cervical canal In a normal woman, after coitus with a fertile man, more than 25 motile sperm (with 2 to 3+ motility)/ high power field (HPF) (x 200) are commonly observed in the exocervical specimen. Ten or more sperm/hpf with directional motility may thus be considered satisfactory. The presence of fewer than five sperm/hpf, particularly when associated with sluggish or circular motion, is an indication of oligo-asthenozoospermia or abnormality of cervical mucus. 23 Beginning 4 hours after coitus, the number of sperm in the exocervical pool gradually decreases. Thus, tests performed at intervals of 4 hours or more after coitus may reveal a fewer number of sperm in the mucus collected from the lower cervical canal. Endocervical Sample. After ejaculation, sperm reach the level of the internal os rapidly. Their numbers increase gradually and reach a peak approximately 2 to 3 hours later. Thereafter, their numbers remain relatively constant for up to 24 hours. At 6 to 8 hours PC, therefore, normally more than 10 sperm/hpf, with adequate motility, should be found. A similar number of sperm is usually detected in a delayed test (8 to 24 hours PC). Interpretation and Causes of a Negative PC Test A negative postcoital test has little clinical value and must be repeated. Controversy continues as to the significance of sperm found in cervical secretion several hours after coitus. Some investigators have suggested that they consist mainly of sperm populations of poor quality which have failed to complete their passage to the uterus. This conclusion is not supported by recent animal and human studies Grant 27 compared the results of postcoital tests and endometrial aspirations in 920 women and found that motile sperm were present in the uteri of HY!o, although they could not be demonstrated in the cervical mucus. An explanation of this finding may be that, in cases of oligospermia or when cervical mucus is

8 124 MOGHISSI February 1976 relatively hostile, the healthier and more vigorous sperm penetrate the cervix and reach the uterine cavity shortly after coitus. Other sperm, which are normally stored in cervical crypts and mucus, do not survive long enough to be detected. Most investigators believe that persistently negative postcoital tests indicate either an abnormality of the mucus or oligo-asthenozoospermia. Hostile Cervical Mucus. The most common cause of a negative PC test is inappropriate timing. Tests performed too early or too late in the cycle may be negative in an otherwise fertile woman. In some women, the test may be positive for only 1 or 2 days during the entire menstrual cycle (Fig. 2). When ovulation cannot be timed with a reasonable degree of accuracy, serial PC tests should be performed. Viscosity and Cell Content. The viscosity of cervical mucus is the greatest barrier to sperm penetration. There is no resistance to sperm migration in thin mucus, but viscous mucus such as that observed during the luteal phase and in pregnant and progestogen-treated women forms an impenetrable barrier. Viscous mucus diluted artificially with normal saline or 5% dextrose is more readily penetrated by sperm than is undiluted mucus. 11 Some relatively viscous samples of cervical mucus have a high degree of surface tension and thus initially appear to be impenetrable by sperm. When these samples are mixed with semen, sperm invasion may occur. This in vitro mixing test (M test) is a useful device for determining the ability of sperm to pene trate the mucus, particularly when it is realized that during coitus such mixing of cervical mucus and semen commonly takes place. The cleanliness of the cervical mucus appears to be a factor favoring sperm migration. Cellular debris and leukocytes impede the progress of sperm in mucus. Severe endocervicitis has been associated with reduced fertility. 28 The precise relationship of mycoplasma infection to the PC test and infertility remains to be determined Effect of ph. Sperm are susceptible to changes in the ph of cervical mucus. Acid mucus immobilizes sperm, whereas alkaline mucus enhances their motility. Excessive alkalinity of cervical mucus (ph greater than 8.5) may also adversely affect the viability of sperm. 31 The optimal ph for sperm migration and survival in cervical mucus is between 7 and This is the ph range of normal, midcycle, cervical mucus. Effect of Immune Antibodies. The role of sperm antibodies in cervical mucus remains uncertain. Although isolated clinical observations and indirect data imply that sperm antibodies in cervical mucus may interfere with fertility, satisfactory demonstration of such a relationship is yet to be established. It is possible that the significant decrease, almost to a negligible level, of immunoglobulins in cervical mucus during the preovulatory phase may be responsible for the absence of interference of antibodies with sperm survival in some cases.33 A high incidence of agglutinating antibody in the cervical mucus of infertile women has recently been reported, but a cause-and-effect relationship between these antibodies and the state of infertility remains to be established. Miscellaneous Causes. Poor coital techniques, vaginismus, and dyspareunia occasionally may be associated with a negative postcoital test. A markedly anteflexed cervix may not come in contact with the semen pool (particularly when semen volume is scanty) and may be a cause of infertility. Vaginal infections and infestations are uncommon causes of negative or abnormal PC tests. Male-Related Causes. A positive postcoital test results only when semen of good quality is deposited in the vagina. Con-

9 Vol. 27, No.2 POSTCOITAL TEST 125 ditions which prevent ejaculation, intromission, or full penetration of the penis into the vagina are associated with a negative PC test. These include premature or retrograde ejaculations, operations on the prostate gland and bladder neck, hypospadias, and impotence. A common cause of a negative postcoital test is abnormal semen. Oligozoospermia, asthenozoospermia, the presence of a large number of morphologically abnormal spermatozoa, and delayed or abnormal liquefaction are known to be associated with inadequate sperm penetration into, or absence of sperm in, the cervical mucus. Uterine Aspiration (Fundal) Test This test is performed as a supplement to the postcoital test. The technique consists of thoroughly cleaning the endocervix, introducing a cannula through the cervix into the uterine cavity, and aspirating the uterine content. The uterine fluid is placed on a glass slide in order to observe the number and motility of sperm per HPF. Many investigators have questioned the value of the test since it is known that the cervical canal cannot be completely cleaned of sperm and that contamination of the endometrial cavity by the cannula commonly occurs. Furthermore, large numbers of spermatozoa are normally located in the endometrial glands, 26 and aspiration and even washing may not remove them from the uterine cavity. Significance of the PC Test Soon after ejaculation, spermatozoa are transferred from the seminal plasma to the female genital tract fluid, in which they are suspended. The migration, survival, and fertilizing potential of spermatozoa depend to a large extent on how they can adapt themselves to this new environment. The postcoital test provides valuable information on sperm-cervical mucus interaction. In Table 1 a selective survey of published reports on postcoital tests is presented. Most investigators believe that PC tests correlate well with in vitro studies. 21 Motile sperm have been observed in samples of cervical mucus obtained from the cervical os and canal 1.5 to 3 minutes after ejaculation. 38 Serial postcoital tests have shown that the increased fluidity and spinnbarkheit of preovulatory cervical mucus coincide with the greater number of motile sperm observed in the mucus samples. 40 After ovulation, the mucus becomes thick, and few, if any, live sperm cells are found in the mucus. Combined postcoital tests and endometrial aspirations have demonstrated that the greatest numbers of sperm in the endometrial cavity are found at, or near, ovulation. Very few are present in the uterus during the luteal or early follicular phases of the cycle. 20 Studies performed by Ahlgren 46 have shown that the number of sperm in the cervical mucus and uterine cavity correlate well with that in the oviducts and pouch of Douglas. The greatest numbers of progressively motile sperm in these locations have been observed in the presence of a mature follicle in the ovaries. A significant decrease in the motile sperm population has been seen when the ovaries contained corpora lutea. However, once the sperm reach the uterine cavity, their further progression to the oviduct may not be influenced by the stage of the ovarian cycle. The presence of sperm in the uterus correlates with the high-sperm density of the cervical mucus, approximately 25 to 41 hours after coitusy The postcoital test has also been correlated with the occurrence ofpregnancy. If the cervical mucus is good and an adequate number of active motile sperm are found at the time of the PC test, the pregnancy rate is significantly greater than it is if poor sperm migration occurs and there is good cervical mucus. 37 Jette and Glass 42 found that the pregnancy rate

10 126 MOGHISSI February 1976 Sims 18 Cary"'~ Grant 27 Author Buxton and Southam 37 TABLE 1. A Selective Survey of Studies on the Postcoital Test Method Random PC tests Random PC tests Random PC tests in 416 women, comparison with fundal test Random PC tests in fertile women Findingsr' Rapid sperm penetration in CM, survival for up to 48 hours Rapid sperm penetration in CM Good correlation with pregnancy; 10% negative PC tests showed sperm in uterus Pregnancy rate higher when CM was of good quality and adequate number of sperm present Sobrero and MacLeod 38 Immediate PC tests Sperm in CM min PC Danezis et al ,577 PC tests in 546 infertile No difference in results within 8 hr PC; women better PC test in those who became pregnant Sujan et al. 40 Serial PC tests Greater number of sperm in CM before than after ovulation Gibor et al.'1 Random PC tests in 505 infertile women Properties of CM not different between women who conceived and those who did not; sperm/hpf greater in fertile group than in infertile group Jette and Glass 42 Random PC tests in 555 infertile Higher pregnancy rate when CM favorable Friberg and Gemzell 43 women Daily PC tests at time of ovulation in 85 women whose ovulations were induced with gonadotropins and when 20 or more sperm/hpf in CM More than 5 and 20 sperm/hpf found in 93 and 64% of women, respectively, hr PC in treatment cycles Kesserii 44 Comparison of PC tests in control Absence of sperm in uteri of infertile group and infertile women Giner et al. 45 Random PC tests in 54 women Lack of correlation between CM quality, no. and degree of motility of sperm in CM, and occurrence of pregnancy Tredway et al. 19 PC tests at various times after in- No. of sperm in CM directly related to sperm "CM, Cervical mucus. semination in 42 women in inseminated semen; maximal no. of sperm in CM 2.5 hr PC in 555 infertile women was significantly higher when there was a favorable cervical mucus and when there were more than 20 sperm/hpf in the mucus, as demonstrated by postcoital tests. Giner et al., 45 however, were unable to demonstrate such a relationship. IN VITRO STUDIES Negative or abnormal PC tests are indications for in vitro cervical mucussperm penetration tests. Two different techniques have been used for in vitro investigation of sperm penetration of cervical mucus: the slide method and the capillary tube system. A good correlation has been found between the results obtained by these techniques. 48 The slide method described by Miller and Kurzrok has been altered to provide quantitative results. 10 In in vitro studies 11 by the slide method, a sharp boundary is observed separating human cervical mucus placed in juxtaposition to semen on a microscope slide (Fig. 5). At the interface, finger-like projections or phalanges of seminal fluid develop within a few minutes and penetrate the mucus. Sperm usually fill these canals before entering the mucus. Most spermatozoa penetrate the apex of the phalangeal canal and then enter the mucus. In most instances, a single spermatozoon appears to lead a column of sperm into the mucus. After the initial resistance has been overcome by the leading spermatozoon, others follow without difficulty. Once in the cervical mucus, the sperm fan out and move at random. Some return to the seminal plasma layer, while most migrate deep into the cervical mucus until they meet resistance from

11 Vol. 27, No.2 POSTCOITAL TEST 127 C.M. FIG. 5. Schematic representation of the in vitro sperm-cervical mucus penetration test by the quantitative slide method. C.M., Cervical mucus; Sp, sperm; I-F, interface; Ph, phalanges;f" first microscopic field; F 2, second field adjacent to F 1 (From Moghissi. 14 ) cellular debris or leukocytes. They then either stop or change direction. Both phalanx and interface formations appear to be physical phenomena resulting from the contact of two biologic fluids of differing viscosities and surface tensions. The capillary tube system is particularly useful for the evaluation of midcycle cervical mucus and for studying the depth of penetration. The slide method is applicable to a variety of cervical mucus samples, including those too viscous or too scanty to be studied by the tube technique. When both in vivo and in vitro tests are negative, normal donor sperm may be tested in vitro against the wife's cervical mucus, and preovulatory mucus obtained from a fertile donor should be tested with the husband's sperm. This cross-match test will determine whether the sperm or cervical mucus is responsible for abnormal results. TREATMENT OF CERVICAL INFERTILITY It is beyond the scope of this essay to discuss the appropriate treatment for all conditions leading to a negative postcoital test. In all cases of infertility / \ associated with an abnormal or negative Sims-Huhner test, the male factor and sperm abnormalities should be investigated thoroughly and excluded. Similarly, anatomical and neoplastic conditions of the cervix should be appropriately treated. If a cervical mucus abnormality is established, the treatment should be directed toward improving the physiochemical properties and sperm receptivity of the mucus. Endocervicitis requires bacteriologic studies and specific antibiotic therapy. Hot cauterization of the endocervix should be avoided. Destruction of the secretory epithelium by other means, such as cold conization and cryosurgery, may have a similarly adverse effect. Viscous mucus is in most instances successfully treated with small doses of estrogen, such as 0.1 to 0.2 mg of stilbestrol or 10 to 20 f.lg of ethinyl estradiovday from day 5 to day 15 of a typical 28-day menstrual cycle. Larger doses of estrogens may inhibit or delay ovulation. A precoital alkaline douche improves the vaginal milieu but has no effect on cervical mucus. Persistent viscosity or acidity of cervical mucus that is unresponsive to estrogen therapy requires intrauterine insemination. For this procedure, at ovulation approximately 0.5 ml of the first portion of a split ejaculate from the husband is slowly injected directly into the uterus, via a polyethylene tube. Occasionally, poor cervical mucus may be a manifestation of inadequate follicular development. Small doses of clomiphene citrate (i.e., 50 mg daily for 4 or 5 days, beginning on day 5 of the cycle), with or without subsequent estrogen as described above, or human menopausal gonadotropin may be useful in improving follicular function, ovarian estrogen output, and cervical mucorrhea. CONCLUSION The postcoital test determines the adequacy of sperm and the receptivity of

12 128 MOGHISSI February 1976 cervical mucus. It is the only test which evaluates the interaction between sperm and the female genital tract fluids. The Sims-Huhner test should be an integral part of an infertility investigation, but it must not be used as a substitute for semen analysis. Since cervical mucus accurately reflects the ovarian cycle, the PC test is a useful indicator of the endocrine preparation of the female reproductive system. It is also an important method for the evaluation of a variety of contraceptive steroids which may act directly or indirectly upon cervical secretion. For best results the test should be properly timed, meticulously performed, and knowledgeably interpreted. REFERENCES 1. MacLeod J, Hotchkiss RS: The distribution of spermatozoa and certain chemical constituents in the human ejaculate. J Urol 48:225, Kellerman AS, WeidJC: Sperm motility and survival in relation to glucose concentration: an in vitro study. Fertil Steril 21:802, Stambaugh R, Buckley J: Comparative studies of the acrosomal enzymes of rabbit, rhesus monkey and human spermatozoa. Bioi Reprod 3:275, Syner FN, Moghissi KS: Proteolytic enzymes in human semen. In Biology of Mammalian Fertilization and Implantation, Edited by KS Moghissi, ESE Hafez. Springfield Ill, Charles C Thomas, 1972, p 3 5. Doehr SA, Moghissi KS: The mucin of human and bovine cervical mucus. In The Biology of the Cervix, Edited by RJ Blandau, KS Moghissi. Chicago, University of Chicago Press, 1973, p Gibbons RA, Mattner P: Some aspects of the chemistry of cervical mucus. In Proceedings of the Fifth World Congress on Fertility and Sterility, Edited by B Westin, N Wiqvist. Amsterdam, Excerpta Medica Foundation, 1967, p Gibbons RA, Mattner P: The chemical and physical characteristics of the cervical secretion and its role in reproductive physiology. In Pathways to Conception, Edited by AI Sherman. Springfield Ill, Charles C Thomas, 1971, p Moghissi KS, Syner FN: Studies on cervical mucus. Fertil Steril 21:234, Neuhaus OW, Moghissi KS: Composition and properties of human cervical mucus. III. A preliminary study of the mucoid component. Fertil Steril13:550, Moghissi KS, Syner FN: The effect of seminal protease and sperm migration through the cervical mucus. Int J Fertil15:43, Moghissi KS, Dabich D, Levine J, Neuhaus OW: Mechanism of sperm migration. Fertil Steril 15:15, Moghissi KS, Syner FN, Evans TN: A composite picture of the menstrual cycle. Am J Obstet Gynecol 114:405, MacDonald RR, Lumley IB: Endocervical ph measured in vivo through the normal menstrual cycle. Obstet Gynecol 35:202, Moghissi KS: Cyclic changes of cervical mucus in normal and progestin-treated women. Fertil Steril 17:663, Sobrero AJ: Sperm migration in the human female. In Proceedings of the Fifth World Congress on Fertility and Sterility, Edited by B Westin, N Wiqvist. Amsterdam, Excerpta Medica Foundation, 1967, p Fluhmann CF: The Cervix Uteri and Its Disease, Third Edition. Philadelphia, WB Saunders Co, Hafez ESE: The comparative anatomy of the mammalian cervix. In The Biology of the Cervix, Edited by RJ Blandau, KS Moghissi. Chicago, University of Chicago Press, 1973, p Sims JM: Uterine Surgery. New York, Wm Woods Co, Tredway DT, Settlage DF, Nakamura RM, Motoshima M, Umezaki CU, Mishell DR: Significance of timing for postcoital evaluation of cervical mucus. Am J Obstet Gynecol121:387, Frenkel DA: Sperm migration and survival in the endometrial cavity. Int J Fertil 6:285, Davajan V, Kunitake G: Fractional in vivo and in vitro examination of post-coital cervical mucus in the human. Fertil Steril 20:197, Moghissi KS: Sperm migration through the human cervix. In Cervical Mucus in Human Reproduction, Edited by M Elstein, KS Moghissi, R Borth. Copenhagen, Scriptor, 1973, p World Health Organization: Cervical mucus, present state of knowledge. In Cervical Mucus in Human Reproduction, Edited by M Elstein, KS Moghissi, R Borth. Copenhagen, Scriptor, 1973, p Mattner PE: Spermatozoa in the genital tract of the ewe. II. Distribution after coitus. Aust J Bioi Sci 16:688, 1963

13 Vol. 27, No.2 POSTCOITAL TEST Mattner PE: The distribution of spermatozoa and leukocytes in the female genital tract in goats and cattle. J Reprod Fertil 17:253, Jaszczak S: Migration of sperm in the cervix and uterus of nonhuman primates. In Cervical Mucus in Human Reproduction, Edited by M Elstein, KS Moghissi, R Borth. Copenhagen, Scriptor, 1973, p Grant A: Cervical hostility. Fertil Steril 9:321, Sobrero AJ: Bacteriological findings in the midcycle endocervical mucus in infertile women. Ann NY Acad Sci 97:591, Horne HW, Kundsin RB, Kosasa TS: The role of mycoplasma infection in human reproductive failure. Fertil Steril 25:380, Patton WC, Taymor ML: An investigation of the relationship between cervical mycoplasma infection, the postcoital test, and infertility (abstr). Fertil Steril 26:211, Tampion D, Gibbons RA: Effect of ph on the swimming rate of bull spermatozoa. J Reprod Fertil 5:249, Carlborg L, Johansson EDB, Gemzell C: Sialic acid content and sperm penetration of cervical mucus in relation to total urinary estrogen excretion and plasma progesterone levels and ovulatory women. Acta Endocrinol (Kbh) 62:721, Schumacher GFB: In Immunological Approaches to Fertility Control. Karolinska Symposia on Research Methods in Reproductive Endocrinology, 7th Symposium, Edited by E Diczfalusy, A Diczfalusy. Geneva, Karolinska, Institutet, 1974, p Shulman S, Patel S, Stamm E: Sperm immunity and infertility: humoral and cellular aspects (abstr). Fertil Steril 26:194, Shulman S, Friedman MR: Antibodies to spermatozoa. V. Antibody activity in human cervical mucus. Am J Obstet Gynecol 122:101, Carey WH:.Sterility diagnosis: the study of sperm cell migration in the female excretions and intetpretation of findings. NY State J Med 30:131, Buxton CL, Southam AL (Editors): Cervical physiology. In Human Infertility. New York, Harper and Row, 1958, p Sobrero AJ, MacLeod J: The immediate postcoital test. Fertil Steril 13:184, Danezis J, Sujan S, Sobrero A: Evaluation of the postcoital test. Fertil Steril 13:559, Sujan S, Danezis J, Sobrero AJ: Sperm migration and cervical mucus studies in individual cycles. J Reprod Fertil 6:87, Gibor Y, Garcia CJ, Cohen MR, Scommegna A: The cyclical changes in the physical properties of the cervical mucus and the results of the postcoital tests. Fertil Steril 21:20, Jette NT, Glass RH: Prognostic value of the postcoital test. Fertil Steril 23:29, Friberg J, Gemzell C: Daily postcoital tests in the conception cycle during treatment of anovulatory women with human gonadotropins. Int J Fertil 17:178, Kesserii E: in vivo sperm penetration and in vitro sperm migration tests. Fertil Steril 24: 584, Giner J, Merino G, Luna J, Aznar R: Evaluation of the Sims-Huhner postcoital test in fertile couples. Fertil Steril 25:145, Ahlgren M: Migration of spermatozoa to the fallopian tubes and the abdominal cavity in women, including some immunological aspects. Student Literatur, Lund, Sweden, 1969, pll 47. Moyer DL, Rimdusit S, Mishell DR: Sperm distribution and degradation in the human female reproductive tract. Obstet Gynecol35:831, Kremer J: The in vitro spermatozoal penetration test. In Fertility Investigation Thesis. Groningen, Netherlands, Drukkerij Van Denderen NV, 1968