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Secretory IgA in cervical mucus
JOURNAL
OF ADOLESCENT
HEALTH
1994;15:423-425
FELLOWSHIP FORUM
ecretory 1.G JANE W. McGRATH, M.D., VICTOR C. STRASBURGER, M.D., AND ALICE H. CUSHING, M.D.
Purpose: Sexually active adolescent girls are uniquely vulnerable to sexually transmitted disease, including cervical cancer and AIDS. Little is known about the development of genital immunity in adolescents. Secretory IgA (s&A) in cervical mucus is an important component of genital immunity. We studied sIgA levels in cervical mucus samples for both adolescent and adult females. Methods: Samples were collected in a univetsitybased adolescent medicine clinic and a university student health center. Participants consisted of 13 sexually active adolescent girls and fourteen adult controls. Samples were collected in the course of routine pelvic exams. All subjects
were at least two years post menarche. Mucus was aspirated directly from the cervical OS. Diluted samples were liquefied with a proteolytic enzyme (bromelain). Secretory IgA levels were measured by radial immunodiffusion using IgA, from pooled human plasma as a standard. Results: Secretory IgA levels for the adolescent group (mean 0.157 g/L SD O.OgOlwere slightly lower than for the adult group mc IEI(0.199 g/L SD 0.1301 although not statistically significant. Conclusions: Cervical s&A levels were comparable between sexually active adolescents and adults.
lescent’s genital tract. The hypothesis of this study was that significant differences exist in immune function between young sexually active adolescents and adult women and that these differences make adolescents more vulnerable to sexually transmitted disease. Immunologic defense of the female genita1 tract occurs primarily at the mucosal surfaces of the lower genital tract. In studies of adult women (1,2) most
of the immunologically
active
cells tire found
in the cervix and vagina. Cervical mucus contains high levels of immunoglobulin and provides a significant barrier to both local and ascending infection (3,4). We analyzed cervical mucus samples for secretory IgA (sIgA) because it is the predominant immunoglobulin in genital secretions and is an important component of humoral immunity in the lower genital tract (5). The intent of this study was to compare sIgA levels in young adoIescents and adult women. Both groups were sexually active and some were using oral contraception at the time of t+ study.
Methods Sexually active adolescent girls have a high incidence of sexually transmitted disease (STDs). The biologic factors which contribute to their risk for disease are largely unexplored. Of particular concern is the local imI; ne system of the female ado-
From the Department of Pediatrics, University of New School ofMedicitle,Albuquerque, New Mexico. Address correspondence
to Jnne W.
Mexico
McGrutk M.D., Depurtmerltof
Pediatrics, University of New Mexico School que, New Mexico 87107. Manuscript accepted january 28.1994.
ofMedirine,
Afbuquer-
Adolescent subjects were recruited from a university-based adolescent health clinic and adult controls were recruited from a university student health center. Informed consent was obtained from each subject in accordance with protocol approved by the Human Research Board. Mucus specimens were collected by direct aspiration at the cervical OS,using an endocervical aspirator (Unimar@ aspirettem). All samples were stored at -70” C. Samples were then diluted with an equal amount of normal saline or norma! saline with 7% bovine serum albumin. To liquify the cervical mucus, all
0 Society for Adolescent Medicine, lW4 Published by Elsevier Science Inc., 655 Avenue of the Americas, New York, NY 10010
423 1054-139x/94/$7.00
JOURNAL OF ADOLESCENT HEALTH Vol. 15, No. 5
MCGRATH ET AL.
424
samples were treated with the enzyme bromelain (6). Bromelain has been used extensively to liquefy cervical mucus to extract soluble proteins such as SIgA. It has been shown to be effective in liquefying cervical mucus and does not significantly alter immunoglobulin structure (7-9). Secretory IgA (IgA,) levels were measured by a radial immunodiffusion (RID) using IgAZ from pooled human plasma as a standard. (The Binding Site Inc) (10,ll). All samples were tested at least two times and calibration curves were determined for each RID plate. Clinical data from patients were collected using a self-administered questionnaire. Thirty-eight subjects were enrolled in the study and twenty-seven subjects completed the study. Eleven subjects were excluded because of known vaginal infection or sexually transmitted disease, sample contamination with blood, or problems with sample preparation. Statistical analysis was performed using an independent samples Student’s t-test to compare the two study groups and a simple linear regression analysis of age since menarche versus sIgA levels.
Results
The mean age of the adolescent group was 15.2 years with a range of 12-17.5 years. All subjects were at least two years post-menarche at the time of the study. The mean age of the adult group was 25 years with a range of 19-40 years. Oral contraceptive pill use was reported by 30% of the adolescent group and 50% of the adult group (Table 1). Secretory IgA levels for the adolescent group (mean = O.l57g/L, SD = 0.080) were slightly lewer than for the adult group (mean = O.l99g/L, SD = 0.130) but the difference‘was n.ot:statistically significant. A simple linear regression analysis of years
Table 1.Characteristics of Characteristics Of The Study Subjects
Age(years) Years since menarche Age at fit
coitus (years)
Number of sexual partners Oral contraceptivepill use
the
Study Subjects
Adolescent
Group
Mean = 15.2 (rargc 12-17.5) Mean = 2.9 (range 2-5) Mean = 14.3 (range 11-17) Mean = 2.5 (range l-9) 4113
since menarche and sIgA levels did not demonstrate a significant correlation (p = 0.06.)
Disctission In this study secretory IgA levels in cervical mucus were lower in adolescents than in adults but not significantly different. This may be a result of study design. Factors which may have inr;luenced the results include: 1) small sample size, 2) age composition of the study groups, 3) sIgA levels resulting from hormonal effects and recent infections, 4) the physical properties oi mucus. The sample size was small-with only 27 samples having been completely analyzed. This small sample size may well have been a factor in the lack of statistically significant results. The age composition of the two study groups was: adolescents ages 17 years and younger; adults ages 18 to 45 years. The adult group was heavily weighted !owards younger subjects: 29% of the adults were either 19 or 20 years old. Infection is another factor known to infhence sIgA levels (12,13). Althouglr the study subjects were tested for commonly diagnosed sexually tmnsmitted diseases and vaginitis, the effects of recent and undetected infection could not be excluded. Cervical mucus is difficult to collect and liquefy. Many potential study subjects were excluded because adequate amounts of cervical mucus could not be obtained at the time. Cervical mucus is not a homogeneous substance; it is very viscous and difficult to work with, The gel properties of mucus impair the quamitative determination of soluble proteins by tra;>ping them within its glycoprotein matrix. Extracting these proteins without causing significant degradation of their structure in the process remains ip challenge. Future research efforts should be directed towards young girls-preferably within one year of menarche. It is in this age group that low levels of cervical sIgA are most likely.
Adult Group Mean = 25 (range 19-40) Mean = 12 (range 6-27) Mean = 17.5 (range 15-19) Mean = 14 (range 2-75) 7/14
References Kutteh WH, Blackwell RE, Gore I-I, et al. Secretcry immune system of the female reproductive tract II. Local immune system in normal and infected fallopian tubes. Rrtil Steril 1990;54f1):51-5. Kutteh WH, Hatch KD, Blackwell RE, et al. Secretory immune system of the female reproductive tract 1. Immunoglobuli and secretory component-containing cells. Obstet Gyneco1 1388;71f1):56-60.
July 1994
SECRETGRY IGA IN CERVICAL MUCUS
42.5
3. Waldman RH, Cruz JM, Rowe DS. lmmunoglobulin levels and antibody to Candida albicans in human cervicovaginal secretions. Clin Exp Immunol1972;10:427-34.
9. lngerslev H, Paulsen F Bromelain for liquefaction of cervical mucus in sperm antibody testing: Its effect on spermagglutinating immunoglobulin G. Fertil Steril 1980;33(1I:61-63.
4. Merriman H, Woods S, Winter C, et al. Secretory IgA antibody in cervicovagina! secretions from women with genital infection due to herpes simplex virus. J Infect Dls 1984;4: 505-10.
10. Cassulis I’, Farhangfar R, DeBari A. Immunoglobulin A subclass distribution in serum from patients with the acquired immunodeficiency syndrome. Clin Biochem 1991;24:227-81.
5. Mestecky J, McGhee JR. Immunoglobulin A fIgA): Molecular and cellular interactions involved in IgA biosynthesis and immune responses. Adv Immunol1987;40:153-65. to 6. Clarke GN. Protocol for the detection of auto-antibodies sperm ceU using immunobead reagent. Bio Rad Bull # 1170 Bio Rad Laboratoties, Richmond, Ca. 7. Cerasaro M, Lenzi A, Massacesi A, et al. Sperm antibodies in infertile women: A comparison of three methods for handling the cervicaI mucus for extraction of immunoglobulins. Int J Fertil 1984;29(3):16&75. 8. Van Kooji R. Solubilization of cervical mucus with bromelain and mechanical means, Int J FertilI984;29f2):81-4.
11. Conley ME, Arbeter A, DougIrs SD. Serum levels of lgA, and IgA, in Children and in Patients with IgA Deficiency. MOL 1mmun011983;20:977-81. 12. Schumacher GFB, Kim MH, Hosseinian AI-I, et al. Immunoglobulins, proteinase inhibitors, albumin, and lysozvme in human cervical mucus. Am J Obstct Gynecol 1977;129(6): 629-36. 13. Waldman RI-I, Cruz JM, Rowe DS Sperm migration-inhibiting antibody in human cervicovaginal secretions. Clin Exp Immunol 1972;12:49-54. 14. Chipperfield E, Evans BA. Etfect of local infection and oral contraception on immunoglobulin levels in cervical mucus. Infect Immunol 1975;11(2):215-21.
OF ADOLESCENT
HEALTH
1994;15:423-425
FELLOWSHIP FORUM
ecretory 1.G JANE W. McGRATH, M.D., VICTOR C. STRASBURGER, M.D., AND ALICE H. CUSHING, M.D.
Purpose: Sexually active adolescent girls are uniquely vulnerable to sexually transmitted disease, including cervical cancer and AIDS. Little is known about the development of genital immunity in adolescents. Secretory IgA (s&A) in cervical mucus is an important component of genital immunity. We studied sIgA levels in cervical mucus samples for both adolescent and adult females. Methods: Samples were collected in a univetsitybased adolescent medicine clinic and a university student health center. Participants consisted of 13 sexually active adolescent girls and fourteen adult controls. Samples were collected in the course of routine pelvic exams. All subjects
were at least two years post menarche. Mucus was aspirated directly from the cervical OS. Diluted samples were liquefied with a proteolytic enzyme (bromelain). Secretory IgA levels were measured by radial immunodiffusion using IgA, from pooled human plasma as a standard. Results: Secretory IgA levels for the adolescent group (mean 0.157 g/L SD O.OgOlwere slightly lower than for the adult group mc IEI(0.199 g/L SD 0.1301 although not statistically significant. Conclusions: Cervical s&A levels were comparable between sexually active adolescents and adults.
lescent’s genital tract. The hypothesis of this study was that significant differences exist in immune function between young sexually active adolescents and adult women and that these differences make adolescents more vulnerable to sexually transmitted disease. Immunologic defense of the female genita1 tract occurs primarily at the mucosal surfaces of the lower genital tract. In studies of adult women (1,2) most
of the immunologically
active
cells tire found
in the cervix and vagina. Cervical mucus contains high levels of immunoglobulin and provides a significant barrier to both local and ascending infection (3,4). We analyzed cervical mucus samples for secretory IgA (sIgA) because it is the predominant immunoglobulin in genital secretions and is an important component of humoral immunity in the lower genital tract (5). The intent of this study was to compare sIgA levels in young adoIescents and adult women. Both groups were sexually active and some were using oral contraception at the time of t+ study.
Methods Sexually active adolescent girls have a high incidence of sexually transmitted disease (STDs). The biologic factors which contribute to their risk for disease are largely unexplored. Of particular concern is the local imI; ne system of the female ado-
From the Department of Pediatrics, University of New School ofMedicitle,Albuquerque, New Mexico. Address correspondence
to Jnne W.
Mexico
McGrutk M.D., Depurtmerltof
Pediatrics, University of New Mexico School que, New Mexico 87107. Manuscript accepted january 28.1994.
ofMedirine,
Afbuquer-
Adolescent subjects were recruited from a university-based adolescent health clinic and adult controls were recruited from a university student health center. Informed consent was obtained from each subject in accordance with protocol approved by the Human Research Board. Mucus specimens were collected by direct aspiration at the cervical OS,using an endocervical aspirator (Unimar@ aspirettem). All samples were stored at -70” C. Samples were then diluted with an equal amount of normal saline or norma! saline with 7% bovine serum albumin. To liquify the cervical mucus, all
0 Society for Adolescent Medicine, lW4 Published by Elsevier Science Inc., 655 Avenue of the Americas, New York, NY 10010
423 1054-139x/94/$7.00
JOURNAL OF ADOLESCENT HEALTH Vol. 15, No. 5
MCGRATH ET AL.
424
samples were treated with the enzyme bromelain (6). Bromelain has been used extensively to liquefy cervical mucus to extract soluble proteins such as SIgA. It has been shown to be effective in liquefying cervical mucus and does not significantly alter immunoglobulin structure (7-9). Secretory IgA (IgA,) levels were measured by a radial immunodiffusion (RID) using IgAZ from pooled human plasma as a standard. (The Binding Site Inc) (10,ll). All samples were tested at least two times and calibration curves were determined for each RID plate. Clinical data from patients were collected using a self-administered questionnaire. Thirty-eight subjects were enrolled in the study and twenty-seven subjects completed the study. Eleven subjects were excluded because of known vaginal infection or sexually transmitted disease, sample contamination with blood, or problems with sample preparation. Statistical analysis was performed using an independent samples Student’s t-test to compare the two study groups and a simple linear regression analysis of age since menarche versus sIgA levels.
Results
The mean age of the adolescent group was 15.2 years with a range of 12-17.5 years. All subjects were at least two years post-menarche at the time of the study. The mean age of the adult group was 25 years with a range of 19-40 years. Oral contraceptive pill use was reported by 30% of the adolescent group and 50% of the adult group (Table 1). Secretory IgA levels for the adolescent group (mean = O.l57g/L, SD = 0.080) were slightly lewer than for the adult group (mean = O.l99g/L, SD = 0.130) but the difference‘was n.ot:statistically significant. A simple linear regression analysis of years
Table 1.Characteristics of Characteristics Of The Study Subjects
Age(years) Years since menarche Age at fit
coitus (years)
Number of sexual partners Oral contraceptivepill use
the
Study Subjects
Adolescent
Group
Mean = 15.2 (rargc 12-17.5) Mean = 2.9 (range 2-5) Mean = 14.3 (range 11-17) Mean = 2.5 (range l-9) 4113
since menarche and sIgA levels did not demonstrate a significant correlation (p = 0.06.)
Disctission In this study secretory IgA levels in cervical mucus were lower in adolescents than in adults but not significantly different. This may be a result of study design. Factors which may have inr;luenced the results include: 1) small sample size, 2) age composition of the study groups, 3) sIgA levels resulting from hormonal effects and recent infections, 4) the physical properties oi mucus. The sample size was small-with only 27 samples having been completely analyzed. This small sample size may well have been a factor in the lack of statistically significant results. The age composition of the two study groups was: adolescents ages 17 years and younger; adults ages 18 to 45 years. The adult group was heavily weighted !owards younger subjects: 29% of the adults were either 19 or 20 years old. Infection is another factor known to infhence sIgA levels (12,13). Althouglr the study subjects were tested for commonly diagnosed sexually tmnsmitted diseases and vaginitis, the effects of recent and undetected infection could not be excluded. Cervical mucus is difficult to collect and liquefy. Many potential study subjects were excluded because adequate amounts of cervical mucus could not be obtained at the time. Cervical mucus is not a homogeneous substance; it is very viscous and difficult to work with, The gel properties of mucus impair the quamitative determination of soluble proteins by tra;>ping them within its glycoprotein matrix. Extracting these proteins without causing significant degradation of their structure in the process remains ip challenge. Future research efforts should be directed towards young girls-preferably within one year of menarche. It is in this age group that low levels of cervical sIgA are most likely.
Adult Group Mean = 25 (range 19-40) Mean = 12 (range 6-27) Mean = 17.5 (range 15-19) Mean = 14 (range 2-75) 7/14
References Kutteh WH, Blackwell RE, Gore I-I, et al. Secretcry immune system of the female reproductive tract II. Local immune system in normal and infected fallopian tubes. Rrtil Steril 1990;54f1):51-5. Kutteh WH, Hatch KD, Blackwell RE, et al. Secretory immune system of the female reproductive tract 1. Immunoglobuli and secretory component-containing cells. Obstet Gyneco1 1388;71f1):56-60.
July 1994
SECRETGRY IGA IN CERVICAL MUCUS
42.5
3. Waldman RH, Cruz JM, Rowe DS. lmmunoglobulin levels and antibody to Candida albicans in human cervicovaginal secretions. Clin Exp Immunol1972;10:427-34.
9. lngerslev H, Paulsen F Bromelain for liquefaction of cervical mucus in sperm antibody testing: Its effect on spermagglutinating immunoglobulin G. Fertil Steril 1980;33(1I:61-63.
4. Merriman H, Woods S, Winter C, et al. Secretory IgA antibody in cervicovagina! secretions from women with genital infection due to herpes simplex virus. J Infect Dls 1984;4: 505-10.
10. Cassulis I’, Farhangfar R, DeBari A. Immunoglobulin A subclass distribution in serum from patients with the acquired immunodeficiency syndrome. Clin Biochem 1991;24:227-81.
5. Mestecky J, McGhee JR. Immunoglobulin A fIgA): Molecular and cellular interactions involved in IgA biosynthesis and immune responses. Adv Immunol1987;40:153-65. to 6. Clarke GN. Protocol for the detection of auto-antibodies sperm ceU using immunobead reagent. Bio Rad Bull # 1170 Bio Rad Laboratoties, Richmond, Ca. 7. Cerasaro M, Lenzi A, Massacesi A, et al. Sperm antibodies in infertile women: A comparison of three methods for handling the cervicaI mucus for extraction of immunoglobulins. Int J Fertil 1984;29(3):16&75. 8. Van Kooji R. Solubilization of cervical mucus with bromelain and mechanical means, Int J FertilI984;29f2):81-4.
11. Conley ME, Arbeter A, DougIrs SD. Serum levels of lgA, and IgA, in Children and in Patients with IgA Deficiency. MOL 1mmun011983;20:977-81. 12. Schumacher GFB, Kim MH, Hosseinian AI-I, et al. Immunoglobulins, proteinase inhibitors, albumin, and lysozvme in human cervical mucus. Am J Obstct Gynecol 1977;129(6): 629-36. 13. Waldman RI-I, Cruz JM, Rowe DS Sperm migration-inhibiting antibody in human cervicovaginal secretions. Clin Exp Immunol 1972;12:49-54. 14. Chipperfield E, Evans BA. Etfect of local infection and oral contraception on immunoglobulin levels in cervical mucus. Infect Immunol 1975;11(2):215-21.