- Email: [email protected]
Temporal relationship between uniplant insertion and changes in cervical mucus
ELSEVIER
Temporal Relationship Between Uniplant Insertion and Changes in Cervical Mucus lone C. Barbosa,* Ulf Ulmstent
Elsimar Coutinho,*
Cristina Hirsch,* O.A. Ladipo,*
This study was undertaken to determine the time required by a single implant containing nomegestrol acetate to affect cervical mucus production and sperm penetration in women. All subjects were investigated and, if necessary, treated for any kind of cervicitis or vaginitis prior to starting cervical mucus study. The subjects had not used hormonal contraception for at least three months prior to investigation. They were counseled to use condoms during this study and also to refrain from intercourse during the period of cervical mucus sampling. Follicular development and endometrial thickness were analyzed by transvaginal sonography. Cervical mucus examination, sperm penetration test, and transvaginal sonography were performed during the control cycle and during the first cycle of Uniplant use. Blood samples were taken for the measurement of estradiol, LH, and progesterone. Cervical mucus and sperm penetration tests were evaluated according to the World Health Organization (WHO) criteria. In the treated cycle, when cervical mucus reached a score of 8-10, Uniplant was inserted, independent of the day of the cycle. Cervical mucus was then collected at 0, 4, 8, 12, 24, 48, and 96 h later until a marked change in volume, consistency, ferning, spinnbarkheit, and cellularity was observed. All samples were also used for sperm penetration test. Preovulatory estradiol and LH peak decreased significantly compared to pre-implant insertion. Progesterone levels were within the normal limit. Cervical mucus and sperm penetration tests were not affected by Vniplant in the first 12 h. Twenty-four hours after Uniplant insertion, cervical mucus and sperm penetration tests were affected in 70.6% of the women. Forty-eight hours after implant insertion, 100% of the women were affected. Follicular rupture occurred in the majority of the women 48 h after implant *Maternidade Climerio de Oiiveira, Federal University of Bahia, Rua do Limoeiro. No. 1, Salvador, 40.055150 Bahia, Brazil, and tthe Department of Obstetrics and Gynecology, Uppsala University, Uppsala, Sweden Name and address for correspondence: Dr. lone C. Barbosa. Maternidade Climerio de Oliveira. Federal University of Bahia, Rua do Limoeiro, No. 1, Salvador, 40.055150 Bahia, Brazil. Tel: 55 71 243 0244; Fax: 55 71 243 9153 Submitted for publication April 26, 1996 Revised June 28, 1996 Accepted for publication July 1, 1996
0 1996 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
Sven Eric Olsson,t
and
insertion. Based on these results, it is possible to conclude that Uniplant can affect estradiol and LH preovulatory peaks and disrupt the process of cervical mucus production and sperm penetration, but it was unable to prevent ovulation when inserted in the preovulatory phase. 0 1996
Elsevier Science Inc. All rights reserved. 1996;54:213-217
CONTRACEPTION
WORDS: Uniplant, cervical mucus, sperm penetration test, transvaginal sonography
KEY
Introduction he quality of cervical mucus (CM) has been recognized as one of several factors determining conception in women.1,2 Moghissi and Marks3 and Gutierrez et a1.4 reported that progestogen-only methods reduce the secretion of CM and cause inhibition of sperm penetration, as determined by in vitro tests. Barbosa et a15j6 found that the levonorgestrelreleasing intrauterine device (IUD, Levo-Nova; Leiras, Turku, Finland) exerted an effect on ovarian function that consisted of disturbances in follicular growth and rupture, and lower plasma levels of Ez, LH, and P. These authors also found that, in the regularly menstruating women, 61.1% of the cycles had good CM, according to the World Health Organization (WHO) criteria.7 Studies with a nomegestrol acetate implant have shown that it affects CM production in all women studied during two years of Uniplant use.’ In these studies with Uniplant, the implant was inserted between days 1 and 5 of the menstrual cycle and CM collection was started on day 8 of the cycles. Exactly when CM production and sperm penetration tests are altered by Uniplant following insertion later on in the first cycle of use is unknown. The aim of this study was to determine the time required by Uniplant to affect CM production and sperm penetration tests once this process has been initiated in the periovulatory phase.
T
ISSN OOlO-7824/96/$15.00 PII SOOlO-7824(96)00191-6
214
Barbosa
Material
et al.
and Methods
Seventeen healthy women with a mean age of 24.62 + 0.99 years, mean weight 56.16 + 2.47 kg, mean height 162.07 + 1.69 cm, mean systolic blood pressure 105.29 f 1.94 mm Hg and mean diastolic blood pressure 65.29 + 1.74 mm Hg, volunteered for this study. All had a history of normal, regular menstrual cycles of a mean of 29.2 -c 0.48 days. They had all been pregnant (mean 1.5 + 0.27 times) and had delivered a mean of 1 .O + 0.14 children. They had not used hormonal contraception or IUDs for at least three months prior to the study. The couples used condoms for contraception during the two cycles of the study. During the period of CM collection, the couples were asked to refrain from having intercourse, even with condoms. All women were asked not to use any prostaglandin synthetase inhibitors for three months prior to the study and during the study period. All women were studied for one cycle before implant insertion and during the cycle when the implant was inserted. Nomegestrol acetate implant (Uniplant, South-toSouth Cooperation in Reproductive Health, Brazil) is a new, long-acting, 19nor-progesterone derivative contraceptive. The implants were handmade from medical-grade dimethylpolysiloxane (SilasticJ tubing, catalog number 602-265, made by Dow Corning, Midland, MI. Segments measuring 39 mm in total length (35 mm of filled length) and 2.4 mm in diameter were used to make the implants. The segments of Silastic tubing were filled with 55 mg (ilO%) of crystalline, finely-ground nomegestrol acetate (3,20-0x0-6methyl- 17-a-acetoxy- 19-norpregna-4,6-diene; Theramex, France) and sealed at both ends with Silastic medical adhesive, type A. Implants were then steam sterilized. Following intracutaneous local anesthesia with 2% procaine, one single capsule was inserted subcutaneously in the gluteal region of the subjects.“” Uniplant
Insertion
Uniplant was inserted in all volunteers when a CM score of 8-10 was obtained, based on the following parameters: volume, consistency, ferning, spinnbarkheit, and cellularity. Uniplant was inserted independent of the day of the cycle. Uniplant was inserted in 15 out of 17 women when CM score was 10, in one woman when CM score was 12, and in another woman when it was 9. Uniplant was inserted in one woman on day 10 of the cycle. Seven women received the implant on day 11, three on day 12, two on day 13, one on day 14, one on day 15, and two on day 18 of the menstrual cycle. The choice of day was based only on the CM score. Venous blood samples were drawn from day 8 of the
Contraception 1996;54:213-217
cycle, every other day, until sonographic evidence of a lO.O-mm follicle and a CM score greater than 5, then every day until sonographic evidence of follicular rupture, and thereafter every other day until the next menstrual bleeding. Cervical Mucus
The external OS was swabbed carefully with dry gauze to avoid contamination. For collection of mucus, the tip of a thin pediatric feeding tube was placed just inside the external OS with an adapted lo-ml syringe, on the same days on which venous blood samples were collected. CM score was evaluated according to the World Health Organization (WHO) criteria.7 The maximum score for CM is 15. A score greater than 10 is indicative of good CM, favoring sperm penetration, and a score less than 10 represents unfavorable CM. The score was derived from the volume, consistency, ferning, spinnbarkheit, and cellularity. CM was evaluated just after collection. The mucus was placed on a slide and allowed to dry. After 2 h, four microscopic fields (HPFj40 x 10) were examined in order to study ferning and cellularity. In the first four women studied, CM was collected at 0,4, 8, 12, and 24 h after implant insertion. Since no changes were observed during the first 12 h, CM from the remaining women in the study was collected at 0, 8, 24, 48, and 96 h as described above. Sperm Penetration
Test
CM was aspirated into a capillary tube. One end of the tube was sealed with modeling clay. The CM was kept at -20°C for a maximum of two months. Sperm penetration in CM was evaluated according to the WHO criteria.’ After all mucus samples were thawed, they were tested in the sperm penetration test with the same sperm sample, which had previously been analyzed and found to be within normal limits for volume, sperm density, morphology, and migration. The open end of the capillary tube was placed into a reservoir containing the semen sample, in a gas incubator at 37°C for one hour. Linear penetration, density of penetration, and motility were assessed as follows: Linear penetration: Depth of penetration = distance (in cm) reached by the most advanced spermatozoa. Density of penetration: The number of sperm in selected areas of the capillary tube (LPF 10 x 10). Motility: The forward motility of the spermatozoa in the upper third of the capillary tube was scored as follows: 0 = no progressive
motility
Contraception 1996;54:213-217
Cervical
1 = 25% of spermatozoa having forward motility 2 = 25-50% of spermatozoa having forward motility 3 = >50% of spermatozoa having forward motility Table 1 illustrates the scoring used. A maximum score of 9 represented an excellent test. The minimum score was 0, representing a negative test. In between, the score of 3 represents a poor test, while 6 represents a good test (Kramer Test). Follicular Development Thickness/texture
and Endometrial
Follicular growth pattern was assessed in all volunteers by transvaginal sonography, scanning both ovaries, using the technique described by Osmers” and Goswany”. The examination started on day 8 of the cycle and was repeated every other day until sonographic evidence of a lo-mm follicle, then every day until evidence of follicular rupture, and thereafter every other day until the onset of the next menstrual bleeding. Endometrial thickness/texture was measured from the proximal to the distal echogenic interface of the junction between the endometrium and the myometrium by an electronic caliper built into the machine. Only the longitudinal section of the uterus was used for measurement of uterine and endometrial thickness. The evaluation of endometrial thickness and texture was performed according to previous studies 13,14 1) The central linear echo representing the endometrial cavity often surrounded by two linear echoes, representing the myoendometrial function; 2) The hypoechogenic layers surrounding the central linear echo representing the growing endometriurn; and 3) The increasing echogenicity starting from the peripheral borders of the myoendometrial echoes, representing differential changes of endometrial layers. Endometrial thickness/texture evaluation was performed each time follicular development was observed.
Statistical
score
Score
0
1
Linear penetration (cm) Density of penetration Motility in the upper third of the capillary tube
0 0
o-2 l-10
0
1
2
3
2-5 1 l-50
l-10 >50
2
3
Users
215
Analysis
For statistical comparison, Student’s t-test for paired groups and Wilcoxon’s non-parametric test were used. Values are shown as mean rt standard error (SE).
Results Mean plasma estradiol level 48 h before ovulation and pre-implant insertion was 674.1 + 89 pmol/l, decreasing to 573.5 + 92 at 24 h and 395.3 + 50.7 at 48 h following implant insertion. The mean plasma estradiol peak in the control cycle was 1087.1 f 95.2 pmol/ 1. This difference was statistically significant (p = 0.0027) compared to the cycle in which Uniplant was inserted (Tables 2 and 3). Mean plasma LH level 48 h before ovulation and pre-implant insertion was 24.4 A 5.2 IU/l, decreasing to 8.9 + 1.2 at 24 h and 5.7 + 1.2 at 48 h following implant insertion. In the control cycle, the mean LH preovulatory peak was 40.4 + 3.6 IU/l. This difference was statistically significant (p = 0.0045) compared to the cycle in which Uniplant was inserted [Table 2). No significant difference (p = 0.055) was observed in progesterone plasma levels when compared to the control cycle. Progesterone levels were lower than the controls, but this difference was not statistically sig2. Hormonal levels and follicular post-implant insertion
Table
Progesterone was determined by radioimmunoassay using a commercial kit from Diagnostic Products 1. Sperm penetration
in Uniplant
Corp., Los Angeles, CA, USA. The sensitivity was 0.09 nmol/l and the interassay coefficient of variation was 6.4%. The normal values were 0.3-89 nmol/l. Estradiol was determined by radioimmunoassay using a commercial kit from Diagnostic Products Corp. The sensitivity was 5.3 pmol/l and the interassay coefficient of variation was 5.5%. The normal values were 146-1468 pmol/l. LH was determined by radioimmunoassay using a commercial kit from Diagnostic Products Corp. The sensitivity was 1.21 II-I/l and the interassay coefficient of variation was 8.3%. The normal values were 15-90 IU/l.
Hormonal Determinations
Table
Mucus
Pre-implant insertion 24 h after implant insertion 48 h after implant insertion *p < 0.05. **p < 0.01. All levels: mean
zt SE.
diameter pre- and
LH Levels IU/l
Estradiol Levels pmol/l
24.4 + 5.2
674.1 zt 89.9
Follicular Diameter mm 18.0 zt 0.5
216
Barbosa
Table
et al.
Contraception 1996;54:213-217
3. Hormonal levels in control and treatment cycles Control Cvcle
Preovulatory estradiol peak (pmol/l) Preovulatory LH peak W/l1 Mid-luteal progesterone peak [nmol/l) Mid-luteal estradiol neak bmolill
1087.1 * 95.2
Treatment 539.4*
* 98.8
40.4 + 3.5
12.0’ f 3.5
53.8 + 2.0
46.6
722.4 +_58.4
882.5
zt 3.4 zt 144.1
‘p < 0.01. All
levels:
mean
* SE.
nificant. In the treated cycle, the mean progesterone level was 43.9 * 4.1 and in the control cycle it was 53.6 + 2.4 nmol/l (Table 3). No statistically significant difference was observed in mid-luteal estradiol peak compared to the control cycle (Table 3). Mean preovulatory CM score for the control cycles was 14.5 + 0.3. Uniplant was inserted when the mean CM score was 10.1 -e 0.2. Eight hours after implant insertion, the mean CM score was 10.1 k 0.2, whereas 24 h after implant insertion, it was 7.3 2 0.8, and 48 h after implant insertion, the mean CM score was 3.0 + 0.6 (Table 4). The mean endometrial thickness in the treated cycle was 11.8 + 0.46 mm. No statistically significant difference was observed when the treated cycles were compared to the control cycles. The mean follicular diameter 48 h before ovulation and pre-implant insertion was 18.0 + 0.5. Twenty-four hours following implant insertion, the mean follicular diameter was 20.0 f 0.6, and 48 h after implant insertion and pre-ovulation, the mean follicular diameter was 19.7 + 0.6 (Table 2). Sperm Penetration Tests For the control group, the mean score for sperm penetration test was 7.2 f 0.4. Uniplant was inserted 4. Cervical mucus score pre- and post-implant insertion Table
Cervical Mucus Score Control Pre-implant insertion 8 h after implant insertion 24 h after implant insertion 48 h after implant insertion
‘p < 0.01. All
scores:
mean
* SE.
when the mean CM score was 10.1 + 0.2. The mean score for the sperm penetration test was 6.6 f 0.6. Eight hours after the implant insertion, the mean score was 6.8 + 0.5. These differences were not statistically significant. Twenty-four hours after implant insertion, the mean score was 3.8 + 0.3, and 48 h after implant insertion, the mean score for sperm penetration was 2.4 + 0.4. These scores were statistically different when compared to the pre-insertion score and to the control group. Of the 17 women studied, one ovulated 24 h after implant insertion, 12 ovulated 48 h after implant insertion, two ovulated 96 h after implant insertion, and one ovulated 6 days after implant insertion. One woman had no follicular rupture (persistent follicle) and showed no increase in progesterone levels.
Discussion The human uterine cervical secretion has an important role for transport, survival, and capacitation of the spermatozoa. The CM changes its characteristics throughout the menstrual cycle following the cyclic changing levels of estradiol and progesterone. At the time of ovulation, the secretory products become abundant, watery, and viscous and the mucin component is formed as long fibrils.15-17 Nomegestrol acetate is a 19-nor-progesterone derivative with potent progestational activity and no androgenicity.8,‘s Uniplant has a potent effect on CM production in women during a period of 24 months of its use.’ In this study, 55 mg of nomegestrol acetate was used in a single Silastic capsule. The release through the Silastic membrane is very stable throughout the duration of use.19 The present study showed that when Uniplant is inserted in the early follicular phase, it is able to affect LH and estradiol preovulatory peaks in all women studied. Progesterone levels were lower than the control cycle, but these differences were not statistically significant. Slight disturbances in follicular growth were observed in 15 out of 17 women. These disturbances consisted of only a small decrease in the volume of the follicle. Sixteen out of 17 cycles were ovulatory according to ultrasonography and progesterone levels. Uniplant affects CM production and sperm penetration 24 h after implant
women studied. Forty-eight
insertion
in 70.6% of the
hours after implant
inser-
tion, Uniplant affected CM production and sperm penetration test in 100% of the women studied.
Based on these results, it is possible to conclude that Uniplant can disrupt the process of CM production and sperm penetration and also affect estradiol
Contraception 1996;54:213-217
and LH preovulatory peaks but, it was unable to prevent ovulation when inserted in the peri-ovulatory phase.
Acknowledgment Supported by the Rockefeller Foundation under the South-to-South Program and by the Family Planning Program of the University of Uppsala, Sweden.
References 1. Jonsson B, Enenoth P, Landgren B-M, Wikborn K. Evaluation of in vitro sperm penetration testing of 176 infertile couples with the use of ejaculate and cervical mucus from donors. Fertil Steril 1986;45:353-6. 2. Jonsson B, Landgren B-M, Enenoth P. Repeated midcycle tests of in vitro spermpenetration (Kramer test) in healthy women during three menstrual cycles. Human Reprod 1989;4:670-3. 3. Moghissi KS, Marks MS. Effects of microdose norgestrel on endogenous gonadotropic and steroid hormones, cervical mucus properties, vaginal cytology and endometrium. Fertil Steril 1971;22:424-34. 4. Gutierrez NA, Giver-Velasquez J, Martinez-Manatou J. Role of cervical mucus in contraception with the continuous chlormadinone acetate method. In: Sobrero A, Lewit S, eds. Advances in Planned Parenthood, Vol. 4. New York:Excerpta Medica Congr. Ser. No. 4, 1969:97. 5. Barbosa I, Bakos 0, Olsson S-E, Odlind V, Johansson EDB. Ovarian function during use of a levonorgestrelreleasing IUD. Contraception 199Oj42:51-65. 6. BarbosaI, Olsson S-E, Odlind V, Goncalves T, Coutinho E. Ovarian function after seven years’ use of a levonorgestrel IUD. Adv Contracep 1995311:85-95. 7. World Health Organization. In: Belsey MA, EliassonR, Gallegos AJ, eds. Laboratory Manual for the Examination of Human and Semen-Cervical Mucus Interaction. Singapore:PressConcern, 1980. 8. BarbosaIC, Coutinho EM, Hirsch C, Ladipo 0, Olsson
Cervical
Mucus
in hiplant
Users
217
S-E, Ulmsten U. Effects of a single contraceptive Silastic implant containing nomegestrol acetate on ovarian function and cervical mucus (CM) production during two years. Fertil Steril 1996j65:724-9. 9. BarbosaIC, Coutinho EM, Athayde C, Ladipo 0, Olsson S-E,Ulmsten U. The effects of nomegestrol acetate subdermal implant (Uniplant) on carbohydrate metabolism, serum lipoproteins and on hepatic function in women. Contraception 1995j52:ll l-4. 10. Coutinho EM. One year contraception with a single subdermal implant containing nomegestrol acetate (Uniplant). Contraception 1993j47:97-105. 11. Osmers R. Transvaginal sonography in endometrial cancer. Ultras Obstet Gynecol 1992j2:23. 12. Goswany RK. Transvaginal ultrasonography: useful for diagnosis.Br Med J 1995j304:331-2. 13. Sakamoto C. Sonographic criteria of phasic changesin human endometrial tissue. Int J Gynecol Obstet 1985; 23:7-12. 14. Yoshimitsu K, Nakamura G, Nakano H. Dating sonographic endometrial images in the normal ovulatory cycle. Int J Gynecol Obstet 1989j28:33-9. 15. Odeblad E. Biophysical techniques of assessingcervical mucus and micro structure of cervical epithelium. In: Elstein M, Moghissi KS, Barth R, eds. Cervical Mucus in Human Reproduction. Copenhagen:Scriptor, 1973; 58-74. 16. Chretian FC. La glaire cervicale III. Roles physiologiques. J Gynecol Obstet Biol Reprod 1974j6:481-8. 17. Chang MC, Hunter RHF. Capacitation of mammalian sperm: Biological and experimental aspects.In: Hamilton DW, Greep RO, eds. Handbook on Physiology, section 7, Endocrinology, Male Reproductive System, Vol. V. Washington:American Physiological Society, 1975j339-51. 18. BarbosaI, Coutinho E, Athayde C, Ladipo OA, Olsson SE, Ulmsten U. Androgen levels in women using a single implant of nomegestrol acetate. Contraception 1996;53:37-40. 19. Ezan E, Benech H, Bucourt R, et al. Enzyme immunoassayfor nomegestrol acetate in human plasma. J Steroid Biochem Molec Biol 1993j46:507-14.
Temporal Relationship Between Uniplant Insertion and Changes in Cervical Mucus lone C. Barbosa,* Ulf Ulmstent
Elsimar Coutinho,*
Cristina Hirsch,* O.A. Ladipo,*
This study was undertaken to determine the time required by a single implant containing nomegestrol acetate to affect cervical mucus production and sperm penetration in women. All subjects were investigated and, if necessary, treated for any kind of cervicitis or vaginitis prior to starting cervical mucus study. The subjects had not used hormonal contraception for at least three months prior to investigation. They were counseled to use condoms during this study and also to refrain from intercourse during the period of cervical mucus sampling. Follicular development and endometrial thickness were analyzed by transvaginal sonography. Cervical mucus examination, sperm penetration test, and transvaginal sonography were performed during the control cycle and during the first cycle of Uniplant use. Blood samples were taken for the measurement of estradiol, LH, and progesterone. Cervical mucus and sperm penetration tests were evaluated according to the World Health Organization (WHO) criteria. In the treated cycle, when cervical mucus reached a score of 8-10, Uniplant was inserted, independent of the day of the cycle. Cervical mucus was then collected at 0, 4, 8, 12, 24, 48, and 96 h later until a marked change in volume, consistency, ferning, spinnbarkheit, and cellularity was observed. All samples were also used for sperm penetration test. Preovulatory estradiol and LH peak decreased significantly compared to pre-implant insertion. Progesterone levels were within the normal limit. Cervical mucus and sperm penetration tests were not affected by Vniplant in the first 12 h. Twenty-four hours after Uniplant insertion, cervical mucus and sperm penetration tests were affected in 70.6% of the women. Forty-eight hours after implant insertion, 100% of the women were affected. Follicular rupture occurred in the majority of the women 48 h after implant *Maternidade Climerio de Oiiveira, Federal University of Bahia, Rua do Limoeiro. No. 1, Salvador, 40.055150 Bahia, Brazil, and tthe Department of Obstetrics and Gynecology, Uppsala University, Uppsala, Sweden Name and address for correspondence: Dr. lone C. Barbosa. Maternidade Climerio de Oliveira. Federal University of Bahia, Rua do Limoeiro, No. 1, Salvador, 40.055150 Bahia, Brazil. Tel: 55 71 243 0244; Fax: 55 71 243 9153 Submitted for publication April 26, 1996 Revised June 28, 1996 Accepted for publication July 1, 1996
0 1996 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
Sven Eric Olsson,t
and
insertion. Based on these results, it is possible to conclude that Uniplant can affect estradiol and LH preovulatory peaks and disrupt the process of cervical mucus production and sperm penetration, but it was unable to prevent ovulation when inserted in the preovulatory phase. 0 1996
Elsevier Science Inc. All rights reserved. 1996;54:213-217
CONTRACEPTION
WORDS: Uniplant, cervical mucus, sperm penetration test, transvaginal sonography
KEY
Introduction he quality of cervical mucus (CM) has been recognized as one of several factors determining conception in women.1,2 Moghissi and Marks3 and Gutierrez et a1.4 reported that progestogen-only methods reduce the secretion of CM and cause inhibition of sperm penetration, as determined by in vitro tests. Barbosa et a15j6 found that the levonorgestrelreleasing intrauterine device (IUD, Levo-Nova; Leiras, Turku, Finland) exerted an effect on ovarian function that consisted of disturbances in follicular growth and rupture, and lower plasma levels of Ez, LH, and P. These authors also found that, in the regularly menstruating women, 61.1% of the cycles had good CM, according to the World Health Organization (WHO) criteria.7 Studies with a nomegestrol acetate implant have shown that it affects CM production in all women studied during two years of Uniplant use.’ In these studies with Uniplant, the implant was inserted between days 1 and 5 of the menstrual cycle and CM collection was started on day 8 of the cycles. Exactly when CM production and sperm penetration tests are altered by Uniplant following insertion later on in the first cycle of use is unknown. The aim of this study was to determine the time required by Uniplant to affect CM production and sperm penetration tests once this process has been initiated in the periovulatory phase.
T
ISSN OOlO-7824/96/$15.00 PII SOOlO-7824(96)00191-6
214
Barbosa
Material
et al.
and Methods
Seventeen healthy women with a mean age of 24.62 + 0.99 years, mean weight 56.16 + 2.47 kg, mean height 162.07 + 1.69 cm, mean systolic blood pressure 105.29 f 1.94 mm Hg and mean diastolic blood pressure 65.29 + 1.74 mm Hg, volunteered for this study. All had a history of normal, regular menstrual cycles of a mean of 29.2 -c 0.48 days. They had all been pregnant (mean 1.5 + 0.27 times) and had delivered a mean of 1 .O + 0.14 children. They had not used hormonal contraception or IUDs for at least three months prior to the study. The couples used condoms for contraception during the two cycles of the study. During the period of CM collection, the couples were asked to refrain from having intercourse, even with condoms. All women were asked not to use any prostaglandin synthetase inhibitors for three months prior to the study and during the study period. All women were studied for one cycle before implant insertion and during the cycle when the implant was inserted. Nomegestrol acetate implant (Uniplant, South-toSouth Cooperation in Reproductive Health, Brazil) is a new, long-acting, 19nor-progesterone derivative contraceptive. The implants were handmade from medical-grade dimethylpolysiloxane (SilasticJ tubing, catalog number 602-265, made by Dow Corning, Midland, MI. Segments measuring 39 mm in total length (35 mm of filled length) and 2.4 mm in diameter were used to make the implants. The segments of Silastic tubing were filled with 55 mg (ilO%) of crystalline, finely-ground nomegestrol acetate (3,20-0x0-6methyl- 17-a-acetoxy- 19-norpregna-4,6-diene; Theramex, France) and sealed at both ends with Silastic medical adhesive, type A. Implants were then steam sterilized. Following intracutaneous local anesthesia with 2% procaine, one single capsule was inserted subcutaneously in the gluteal region of the subjects.“” Uniplant
Insertion
Uniplant was inserted in all volunteers when a CM score of 8-10 was obtained, based on the following parameters: volume, consistency, ferning, spinnbarkheit, and cellularity. Uniplant was inserted independent of the day of the cycle. Uniplant was inserted in 15 out of 17 women when CM score was 10, in one woman when CM score was 12, and in another woman when it was 9. Uniplant was inserted in one woman on day 10 of the cycle. Seven women received the implant on day 11, three on day 12, two on day 13, one on day 14, one on day 15, and two on day 18 of the menstrual cycle. The choice of day was based only on the CM score. Venous blood samples were drawn from day 8 of the
Contraception 1996;54:213-217
cycle, every other day, until sonographic evidence of a lO.O-mm follicle and a CM score greater than 5, then every day until sonographic evidence of follicular rupture, and thereafter every other day until the next menstrual bleeding. Cervical Mucus
The external OS was swabbed carefully with dry gauze to avoid contamination. For collection of mucus, the tip of a thin pediatric feeding tube was placed just inside the external OS with an adapted lo-ml syringe, on the same days on which venous blood samples were collected. CM score was evaluated according to the World Health Organization (WHO) criteria.7 The maximum score for CM is 15. A score greater than 10 is indicative of good CM, favoring sperm penetration, and a score less than 10 represents unfavorable CM. The score was derived from the volume, consistency, ferning, spinnbarkheit, and cellularity. CM was evaluated just after collection. The mucus was placed on a slide and allowed to dry. After 2 h, four microscopic fields (HPFj40 x 10) were examined in order to study ferning and cellularity. In the first four women studied, CM was collected at 0,4, 8, 12, and 24 h after implant insertion. Since no changes were observed during the first 12 h, CM from the remaining women in the study was collected at 0, 8, 24, 48, and 96 h as described above. Sperm Penetration
Test
CM was aspirated into a capillary tube. One end of the tube was sealed with modeling clay. The CM was kept at -20°C for a maximum of two months. Sperm penetration in CM was evaluated according to the WHO criteria.’ After all mucus samples were thawed, they were tested in the sperm penetration test with the same sperm sample, which had previously been analyzed and found to be within normal limits for volume, sperm density, morphology, and migration. The open end of the capillary tube was placed into a reservoir containing the semen sample, in a gas incubator at 37°C for one hour. Linear penetration, density of penetration, and motility were assessed as follows: Linear penetration: Depth of penetration = distance (in cm) reached by the most advanced spermatozoa. Density of penetration: The number of sperm in selected areas of the capillary tube (LPF 10 x 10). Motility: The forward motility of the spermatozoa in the upper third of the capillary tube was scored as follows: 0 = no progressive
motility
Contraception 1996;54:213-217
Cervical
1 = 25% of spermatozoa having forward motility 2 = 25-50% of spermatozoa having forward motility 3 = >50% of spermatozoa having forward motility Table 1 illustrates the scoring used. A maximum score of 9 represented an excellent test. The minimum score was 0, representing a negative test. In between, the score of 3 represents a poor test, while 6 represents a good test (Kramer Test). Follicular Development Thickness/texture
and Endometrial
Follicular growth pattern was assessed in all volunteers by transvaginal sonography, scanning both ovaries, using the technique described by Osmers” and Goswany”. The examination started on day 8 of the cycle and was repeated every other day until sonographic evidence of a lo-mm follicle, then every day until evidence of follicular rupture, and thereafter every other day until the onset of the next menstrual bleeding. Endometrial thickness/texture was measured from the proximal to the distal echogenic interface of the junction between the endometrium and the myometrium by an electronic caliper built into the machine. Only the longitudinal section of the uterus was used for measurement of uterine and endometrial thickness. The evaluation of endometrial thickness and texture was performed according to previous studies 13,14 1) The central linear echo representing the endometrial cavity often surrounded by two linear echoes, representing the myoendometrial function; 2) The hypoechogenic layers surrounding the central linear echo representing the growing endometriurn; and 3) The increasing echogenicity starting from the peripheral borders of the myoendometrial echoes, representing differential changes of endometrial layers. Endometrial thickness/texture evaluation was performed each time follicular development was observed.
Statistical
score
Score
0
1
Linear penetration (cm) Density of penetration Motility in the upper third of the capillary tube
0 0
o-2 l-10
0
1
2
3
2-5 1 l-50
l-10 >50
2
3
Users
215
Analysis
For statistical comparison, Student’s t-test for paired groups and Wilcoxon’s non-parametric test were used. Values are shown as mean rt standard error (SE).
Results Mean plasma estradiol level 48 h before ovulation and pre-implant insertion was 674.1 + 89 pmol/l, decreasing to 573.5 + 92 at 24 h and 395.3 + 50.7 at 48 h following implant insertion. The mean plasma estradiol peak in the control cycle was 1087.1 f 95.2 pmol/ 1. This difference was statistically significant (p = 0.0027) compared to the cycle in which Uniplant was inserted (Tables 2 and 3). Mean plasma LH level 48 h before ovulation and pre-implant insertion was 24.4 A 5.2 IU/l, decreasing to 8.9 + 1.2 at 24 h and 5.7 + 1.2 at 48 h following implant insertion. In the control cycle, the mean LH preovulatory peak was 40.4 + 3.6 IU/l. This difference was statistically significant (p = 0.0045) compared to the cycle in which Uniplant was inserted [Table 2). No significant difference (p = 0.055) was observed in progesterone plasma levels when compared to the control cycle. Progesterone levels were lower than the controls, but this difference was not statistically sig2. Hormonal levels and follicular post-implant insertion
Table
Progesterone was determined by radioimmunoassay using a commercial kit from Diagnostic Products 1. Sperm penetration
in Uniplant
Corp., Los Angeles, CA, USA. The sensitivity was 0.09 nmol/l and the interassay coefficient of variation was 6.4%. The normal values were 0.3-89 nmol/l. Estradiol was determined by radioimmunoassay using a commercial kit from Diagnostic Products Corp. The sensitivity was 5.3 pmol/l and the interassay coefficient of variation was 5.5%. The normal values were 146-1468 pmol/l. LH was determined by radioimmunoassay using a commercial kit from Diagnostic Products Corp. The sensitivity was 1.21 II-I/l and the interassay coefficient of variation was 8.3%. The normal values were 15-90 IU/l.
Hormonal Determinations
Table
Mucus
Pre-implant insertion 24 h after implant insertion 48 h after implant insertion *p < 0.05. **p < 0.01. All levels: mean
zt SE.
diameter pre- and
LH Levels IU/l
Estradiol Levels pmol/l
24.4 + 5.2
674.1 zt 89.9
Follicular Diameter mm 18.0 zt 0.5
216
Barbosa
Table
et al.
Contraception 1996;54:213-217
3. Hormonal levels in control and treatment cycles Control Cvcle
Preovulatory estradiol peak (pmol/l) Preovulatory LH peak W/l1 Mid-luteal progesterone peak [nmol/l) Mid-luteal estradiol neak bmolill
1087.1 * 95.2
Treatment 539.4*
* 98.8
40.4 + 3.5
12.0’ f 3.5
53.8 + 2.0
46.6
722.4 +_58.4
882.5
zt 3.4 zt 144.1
‘p < 0.01. All
levels:
mean
* SE.
nificant. In the treated cycle, the mean progesterone level was 43.9 * 4.1 and in the control cycle it was 53.6 + 2.4 nmol/l (Table 3). No statistically significant difference was observed in mid-luteal estradiol peak compared to the control cycle (Table 3). Mean preovulatory CM score for the control cycles was 14.5 + 0.3. Uniplant was inserted when the mean CM score was 10.1 -e 0.2. Eight hours after implant insertion, the mean CM score was 10.1 k 0.2, whereas 24 h after implant insertion, it was 7.3 2 0.8, and 48 h after implant insertion, the mean CM score was 3.0 + 0.6 (Table 4). The mean endometrial thickness in the treated cycle was 11.8 + 0.46 mm. No statistically significant difference was observed when the treated cycles were compared to the control cycles. The mean follicular diameter 48 h before ovulation and pre-implant insertion was 18.0 + 0.5. Twenty-four hours following implant insertion, the mean follicular diameter was 20.0 f 0.6, and 48 h after implant insertion and pre-ovulation, the mean follicular diameter was 19.7 + 0.6 (Table 2). Sperm Penetration Tests For the control group, the mean score for sperm penetration test was 7.2 f 0.4. Uniplant was inserted 4. Cervical mucus score pre- and post-implant insertion Table
Cervical Mucus Score Control Pre-implant insertion 8 h after implant insertion 24 h after implant insertion 48 h after implant insertion
‘p < 0.01. All
scores:
mean
* SE.
when the mean CM score was 10.1 + 0.2. The mean score for the sperm penetration test was 6.6 f 0.6. Eight hours after the implant insertion, the mean score was 6.8 + 0.5. These differences were not statistically significant. Twenty-four hours after implant insertion, the mean score was 3.8 + 0.3, and 48 h after implant insertion, the mean score for sperm penetration was 2.4 + 0.4. These scores were statistically different when compared to the pre-insertion score and to the control group. Of the 17 women studied, one ovulated 24 h after implant insertion, 12 ovulated 48 h after implant insertion, two ovulated 96 h after implant insertion, and one ovulated 6 days after implant insertion. One woman had no follicular rupture (persistent follicle) and showed no increase in progesterone levels.
Discussion The human uterine cervical secretion has an important role for transport, survival, and capacitation of the spermatozoa. The CM changes its characteristics throughout the menstrual cycle following the cyclic changing levels of estradiol and progesterone. At the time of ovulation, the secretory products become abundant, watery, and viscous and the mucin component is formed as long fibrils.15-17 Nomegestrol acetate is a 19-nor-progesterone derivative with potent progestational activity and no androgenicity.8,‘s Uniplant has a potent effect on CM production in women during a period of 24 months of its use.’ In this study, 55 mg of nomegestrol acetate was used in a single Silastic capsule. The release through the Silastic membrane is very stable throughout the duration of use.19 The present study showed that when Uniplant is inserted in the early follicular phase, it is able to affect LH and estradiol preovulatory peaks in all women studied. Progesterone levels were lower than the control cycle, but these differences were not statistically significant. Slight disturbances in follicular growth were observed in 15 out of 17 women. These disturbances consisted of only a small decrease in the volume of the follicle. Sixteen out of 17 cycles were ovulatory according to ultrasonography and progesterone levels. Uniplant affects CM production and sperm penetration 24 h after implant
women studied. Forty-eight
insertion
in 70.6% of the
hours after implant
inser-
tion, Uniplant affected CM production and sperm penetration test in 100% of the women studied.
Based on these results, it is possible to conclude that Uniplant can disrupt the process of CM production and sperm penetration and also affect estradiol
Contraception 1996;54:213-217
and LH preovulatory peaks but, it was unable to prevent ovulation when inserted in the peri-ovulatory phase.
Acknowledgment Supported by the Rockefeller Foundation under the South-to-South Program and by the Family Planning Program of the University of Uppsala, Sweden.
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S-E, Ulmsten U. Effects of a single contraceptive Silastic implant containing nomegestrol acetate on ovarian function and cervical mucus (CM) production during two years. Fertil Steril 1996j65:724-9. 9. BarbosaIC, Coutinho EM, Athayde C, Ladipo 0, Olsson S-E,Ulmsten U. The effects of nomegestrol acetate subdermal implant (Uniplant) on carbohydrate metabolism, serum lipoproteins and on hepatic function in women. Contraception 1995j52:ll l-4. 10. Coutinho EM. One year contraception with a single subdermal implant containing nomegestrol acetate (Uniplant). Contraception 1993j47:97-105. 11. Osmers R. Transvaginal sonography in endometrial cancer. Ultras Obstet Gynecol 1992j2:23. 12. Goswany RK. Transvaginal ultrasonography: useful for diagnosis.Br Med J 1995j304:331-2. 13. Sakamoto C. Sonographic criteria of phasic changesin human endometrial tissue. Int J Gynecol Obstet 1985; 23:7-12. 14. Yoshimitsu K, Nakamura G, Nakano H. Dating sonographic endometrial images in the normal ovulatory cycle. Int J Gynecol Obstet 1989j28:33-9. 15. Odeblad E. Biophysical techniques of assessingcervical mucus and micro structure of cervical epithelium. In: Elstein M, Moghissi KS, Barth R, eds. Cervical Mucus in Human Reproduction. Copenhagen:Scriptor, 1973; 58-74. 16. Chretian FC. La glaire cervicale III. Roles physiologiques. J Gynecol Obstet Biol Reprod 1974j6:481-8. 17. Chang MC, Hunter RHF. Capacitation of mammalian sperm: Biological and experimental aspects.In: Hamilton DW, Greep RO, eds. Handbook on Physiology, section 7, Endocrinology, Male Reproductive System, Vol. V. Washington:American Physiological Society, 1975j339-51. 18. BarbosaI, Coutinho E, Athayde C, Ladipo OA, Olsson SE, Ulmsten U. Androgen levels in women using a single implant of nomegestrol acetate. Contraception 1996;53:37-40. 19. Ezan E, Benech H, Bucourt R, et al. Enzyme immunoassayfor nomegestrol acetate in human plasma. J Steroid Biochem Molec Biol 1993j46:507-14.