7 combined oral contraceptives

Contraception 86 (2012) 710 – 715

Original research article

Comparison of cervical mucus of 24/4 vs. 21/7 combined oral contraceptives☆ Rachel Steward⁎, Alexander Melamed, Anna Granat, Daniel R. Mishell Jr. Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA Received 19 April 2012; revised 27 April 2012; accepted 3 May 2012

Abstract Background: Few studies have examined the action of combined oral contraceptives (COCs) on cervical mucus. We hypothesized that midcycle cervical mucus of women taking COCs is of poor quality when compared to their own midcycle mucus prior to initiating COCs. We sought to compare the effect upon quality and sperm penetration of the cervical mucus on the last hormone-free day with a 24/4 regimen to a 21/7 regimen. Methods: This is an open-label, investigator-blinded, randomized, controlled, crossover equivalency study. All subjects received, in random order, 2 months of a 21/7 regimen and 2 months of a 24/4 regimen, each containing 20 mcg ethinyl estradiol and 1 mg norethindrone acetate. Analysis of cervical mucus quality (CMQ) and sperm penetration took place midcycle and on the last day of the hormone-free interval during the second month of each COC treatment. Results: From April 2010 to November 2011, 18 subjects completed all study visits. Mean midcycle CMQ was poor (mean CMQ=1) and did not differ between 24/4 and 21/7 regimens (p=.92). On the last day of the pill-free interval, the quality and sperm penetration were poor with both regimens. Conclusion: This study indicates that thickening of cervical mucus is a major mechanism of contraceptive action of COCs and that both 21/7 and 24/4 regimens result in poor quality and impenetrable mucus on the last day of the pill-free interval. © 2012 Elsevier Inc. All rights reserved. Keywords: Cervical mucus; Oral contraceptives; Sperm penetration test

1. Introduction The main contraceptive effect of combined oral contraceptives (COCs) is inhibition of the midcycle lutenizing hormone (LH) surge to prevent ovulation. However, several studies have shown that the percentage of ovulatory cycles in women using low-dose COCs ranges between 1.5% and 16.8% [1–8]. With this high rate of ovulatory cycles in women taking COCs, we would expect the pregnancy rate with COC use to be much higher than the perfect use failure rate of 0.3% [9] were there not other effective mechanisms of contraceptive action in addition to ovulation inhibition. Another potential mechanism of contraceptive action is the suppression of follicle-stimulating hormone secretion ☆

Funding for this study was provided by an anonymous donor. ⁎ Corresponding author. Tel.: + 1 646 417 1685(Cell), 213 919 9864(Pager); fax: +1 323 226 5046. E-mail address: [email protected] (R. Steward). 0010-7824/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.contraception.2012.05.004

during the follicular phase of the cycle, thereby preventing follicular maturation; however, follicular development has been shown to occur in 23%–90% of cycles in women using COCs [1,5,9]. There are also many progestin-related mechanisms that likely contribute to the overall efficacy of the combined contraceptives, such as thickening of cervical mucus, impairment of tubal mobility and peristalsis, and effects on the endometrial lining, making it less suitable for implantation. It is known that sperm transport from the vagina to the oviducts is greatly dependent on the properties of human cervical mucus, including mucus quantity, thickness and hydration [10]. Beginning at approximately the ninth day of an ovulatory menstrual cycle, increasing estradiol levels cause an increase in the amount of cervical mucus [11]. The mucus quality changes, it becomes thin and watery, and it allows sperm penetration from the endocervix into the endometrial cavity. The effect of sperm penetrability increases and reaches a peak just prior to ovulation.

R. Steward et al. / Contraception 86 (2012) 710–715

Progesterone is then secreted from the corpus luteum and causes the cervical mucus to become scant in amount, thick, opaque and unfavorable to sperm penetration [12]. Prior studies have examined the changes in cervical mucus quality (CMQ) and sperm penetrability in women using progestin-only oral contraceptives. Moghissi et al. [13] demonstrated that microdose norgestrel (75 mcg daily) causes alterations of physical and chemical properties of cervical mucus such that it becomes highly viscous, cellular and scanty; exhibits reduced ferning and spinbarkeit; and inhibits sperm transport. Barbosa et al. [14] examined time to contraceptive effectiveness in users of the single implant containing 55 mg nomegestrol acetate. Cervical mucus and sperm penetration tests were altered in all subjects within 48 h after insertion of the implant. Another study of the same design enrolled 30 women receiving an injection of 150 mg depot medroxyprogesterone acetate (DMPA) and measured the same parameters of cervical mucus changes [15]. This study showed all subjects to have CMQ scores of 0, as determined by the World Health Organization (WHO) criteria, and sperm penetrability test sufficient to prevent pregnancy by day 7 after the injection. Lewis et al. [16] demonstrated that midcycle mucus of users of the levonorgestrel intrauterine system is of poor quality and prevents endocervical sperm transport in vitro. Few studies have examined CMQ in users of COC regimens. One study published in 1976 by Elstein et al. [17] indicated that contraceptive action of combined low-dose oral contraceptive is mediated through suppression of ovulation and by rendering cervical mucus impenetrable to sperm. Another randomized trial of two low-dose COCs by Winfried et al. [18] found the quality and quantity of cervical mucus to be minimal in the majority of women during treatment cycles. Another topic that this study aims to address is shortening of the pill-free interval with COCs. A 24/4-day regimen of norethindrone acetate 1 mg/20 mcg ethinyl estradiol was approved for marketing in 2006. This agent has a cumulative pregnancy incidence of 0.9% during the first six cycles of use and a Pearl Index of 1.79 per 100 women-years in women ≤35 years old [19]. In addition to a theoretical improvement in efficacy, a shorter hormone-free interval also has the benefits of decreasing symptoms such as pelvic pain, headache, breast tenderness and bloating/swelling, which occur more frequently during this interval than when active pills are ingested [20,21]. Recently, Dinger et al. [22] published a large US cohort study that demonstrated higher contraceptive effectiveness with a 24-day oral contraceptive regimen compared with the 21-day regimen.

2. Material and methods This was an open-label, investigator-blinded, randomized, controlled, crossover equivalency study comparing the in vitro sperm penetrability and quality of cervical mucus

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using WHO grading criteria on the last hormone-free day of women assigned to one of two groups. Healthy, reproductive-aged women with regular menses who desired initiation of oral contraceptives were enrolled for participation in this study. They were randomized by a computer-generated system to receive an oral contraceptive containing 20 mcg ethinyl estradiol and 1 mg norethindrone acetate either in a 24/4-day regimen or a 21/7-day regimen and started according to “Quick Start” protocol [23]. Inclusion criteria were healthy women aged 18–39 years desiring to initiate COCs. Exclusion criteria were any contraindications to combined hormonal contraception including pregnancy, breastfeeding, liver disease, vascular or uncontrolled metabolic disorders, smoking greater than 15 cigarettes at age 35 or older, body mass index greater than or equal to 40, migraine headaches with aura or untreated cervical dysplasia. Women were excluded if they had used steroid hormone or intrauterine contraception within 3 months prior to study enrollment or 6 months prior in the case of DMPA. Those currently breastfeeding or patients less than 3 months postpartum of a term pregnancy (or within 6 weeks of a first-trimester loss or termination) were also excluded, as were women who would not refrain from intercourse or the use of vaginal douches during the study period requiring cervical mucus assessment. This study was conducted on women in a primarily underserved population. Our informed consents were in both Spanish and English. The study was approved by the University of Southern California Health Science Campus Institutional Review Board. 2.1. Laboratory measures As described by Lewis et al. [16], the tests used to determine mucus quality and penetration were the WHO cervical mucus criteria and the sperm–cervical mucus penetration test detailed below. 2.1.1. Collection of cervical mucus In all subjects, the cervix was exposed using an unlubricated speculum, and the ectocervix was cleansed of debris with a large dry sterile swab. Cervical mucus was then obtained using a specialized endocervical aspirator (Unimar Aspirette, Cooper Surgical, Trumbull, CT, USA). Ring forceps or a cytobrush was used to assist in collecting thick or densely adherent endocervical mucus. Mucus was transported in the collection device to a laboratory where analysis began within 30 min of collection. 2.1.2. Cervical mucus analysis Cervical mucus was grossly and microscopically examined by an investigator blinded to subjects' hormonal status to determine its volume, consistency, cellularity, spinnbarkeit and ferning, as described in the WHO Laboratory Manual for the Examination of Human Semen and Sperm– Cervical Mucus Interaction [24]. A CMQ grading score of 10 or greater of 15 total points favored sperm penetration and

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was considered “good.” A cervical mucus score of less than 10 was considered “poor.” The absence of sperm was confirmed in all samples prior to testing. 2.1.3. Sperm–cervical mucus penetration test To evaluate mucus penetrability by sperm, an in vitro sperm penetration test, first described by Kremer [25] and described in detail by Eggert-Kruse et al. [26], was performed using fresh cervical mucus. The following criteria were examined: migration distance of the foremost spermatozoon, density of penetration using an average of the total number of live sperm at each centimeter between 1 and 5 cm, and the quality of motility at the distal end of the capillary tube. This test was performed at both 2 and 6 h after mucus incubation at 37°C by the principal investigator (PI) who was blinded to the COC randomization. Each parameter was graded on a scale from 0 to 3 and summarized as a total score of 9, according to a scoring system validated in previous studies by correlation with subsequent pregnancies [26,27]. A sperm–cervical mucus penetration score of 9 was “excellent,” a score of 7 or 8 was “good,” and a score less than 7 was considered “poor.” 2.1.4. Semen analysis Frozen semen from a single donor with proven fertility was obtained from a commercial sperm bank (California Cryobank, Los Angeles, CA, USA) with documentation of prethaw sperm count, motility and morphology sufficient for fertilization. Vials were defrosted, and sperm count and percent motility were determined on every vial prior to use by a trained investigator whose techniques were validated by a consulting andrologist and pathologist. Sperm concentration and percent motility were considered to be adequate for testing according to threshold values published in a prior investigation [28]. The concentration and motility of each semen sample were reanalyzed postthaw by the PI, prior to use, to ensure that these parameters remained within normal limits. 2.2. Study protocol If an initial telephone or in-person screening deemed that a woman met initial inclusion criteria, she was invited to present on day 12 of her cycle for a screening visit which included informed consent, provision of screening ID number, collection of demographic information, a complete history and physical examination, gynecological exam and baseline laboratory testing (Pap, APTIMA® collection of cervical secretions for polymerase chain reaction of gonorrhea and chlamydia, and microscopic visualization of vaginal secretions), and cervical mucus evaluation. If all screening tests returned negative and the subject demonstrated good CMQ (WHO score ≥ 10), she was enrolled in the study and given a study ID number. If the cervical mucus she provided was not adequate, she returned in two days (day 14 of her cycle). If, on her second collection day, the mucus was still not sufficient,

she returned in two days (day 16) for a third collection. If, after three mucus collections, her mucus was still not adequate, she was excluded from further participation. Subjects with vaginitis or cervicitis were treated and offered return for study enrollment in 2–4 weeks if test of cure was negative. Once a subject was enrolled, she was started that day on either the 24/4 vs. a 21/7 regimen via a computer-generated block-randomized scheme as dispensed by the pharmacy according to the “Quick Start” protocol [22]. On the 14th day of the second month, the subject presented for her first cervical mucus analysis. This visit occurred on day 12 to day 16 of the menstrual cycle. The visit included a gynecological exam with endocervical mucus sampling using a specialized collection device. The Kremer Sperm Cervical Mucus Penetration Test was used to determine the Sperm Penetration Meter Score (SPMS). After completing two months of the COC, the patient returned on the last day of her hormone-free interval for cervical mucus sampling. If the subject did not present for cervical mucus evaluation, she was given the option of continuing the same pill for an additional month. If a subject did not present for her visits during the third month, she was discontinued from further participation. After 2 completed months of the first regimen, each subject was then switched to the group that she was not originally assigned and began taking the second regimen. Midcycle (day 12 to day 16) of her fourth month, the subject presented for her third cervical mucus sampling. After completing 2 months of the second regimen of COC use, the patient returned on the last day of her hormone-free interval for her final cervical mucus sampling. Each subject was compensated $30 for the screening visit and $20 for each visit thereafter until enrollment. After enrollment, each subject was compensated $40 for each visit. 2.3. Statistical methods Equivalence was defined as mean or median difference in SPMS between + 1 and − 1 unit such that the 95% confidence interval (CI) does not cross these bounds. To establish equivalence in SPMS within a limit of 1 unit at a significance of .05 and a power of 80%, 20 subjects needed to complete the study. This crossover study was analyzed with single-stage analysis, assuming no carryover effects [29]. For normally distributed variables drawn from independent samples, differences in mean were assessed with a two-sample t test, while within-subject differences were tested using the paired t test. For nonnormally distributed variable, the analogous Wilcoxon signed-rank and rank-sum tests were used. Differences among proportions were investigated with the McNemar test with exact binomial probability calculations. Data analysis employed SAS 9.2 (SAS Institute Inc., Cary, NC, USA) and Excel 2003 (Microsoft Corp., Redmond, WA, USA).

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3. Results

Table 1 Baseline characteristics of subjects completing the study (n=18)

Of the 52 subjects screened between April 2010 to November 2011, 32 subjects were enrolled. Of the subjects enrolled, 14 withdrew consent or were discontinued. Eighteen subjects completed all study visits, taking both regimens of the crossover design. Of the 20 subjects screened but not enrolled, 11 were ineligible and 9 were lost to follow-up during the screening process. Of the subjects who were ineligible, seven subjects did not have adequate mucus (WHO ≥ 10), two subjects had cervical dysplasia, one had medical contraindications to estrogen, and one was pregnant upon presentation for mucus analysis. Regarding the 14 subjects who were enrolled but subsequently discontinued, 2 subjects became pregnant on study drug, 11 subjects withdrew consent for inability to comply with the study drug regimen, and 3 subjects were completely lost to follow-up despite rigorous attempts to make contact by the research coordinator and PI (Fig. 1). Baseline characteristics of subjects who completed the trial are illustrated in Table 1. Enrolled study participants had adequate (WHO ≥ 10) quality mucus at baseline (mean CMQ=11.9, 95% CI 11.3–12.5). There were no significant differences in age (p=.23), gravidity (p=.43), parity (p=.78), body mass index (0.57) or ethnicity (p=.91) between subjects randomized to either treatment sequence. As illustrated in Table 2, there was no significant difference in the quality of midcycle cervical mucus obtained on the 21/7 and 24/4 regimens [median CMQ=1

Age, median (IQR) Gravidity, median (IQR) Parity, median (IQR) Body mass index, mean (SD) Race/ethnicity, count (%) Asian Black Hispanic Other White Pretreatment midcycle CMQ, median (IQR)

22 (21–27) 1 (0–1) 0 (0–1) 26.6 (4.7) 2 (11) 5 (28) 6 (33) 2 (11) 3 (17) 12 (11–13)

for both; interquartile range (IQR) 0–3 and 0–2, respectively; p=.92]. The quality of mucus collected on the last hormone-free day of 21/7 and 24/4 regimens did not differ significantly either (median CMQ=2 and 4, respectively; IQR 0–5 and 3–5, respectively; p=.1). On the 21/7 and 24/4 COC regimens, cervical mucus obtained at midcycle, and on the final hormone-free day demonstrated inadequate (b10) median CMQ scores (pb.001 at both time periods). At midcycle, no study participants had adequate CMQ on either regimen. On the final hormone-free day, 1/18 subjects had adequate CMQ while receiving the 21/7 regimen, and none had adequate CMQ while receiving the 24/4 regimen (p=.1). In this study, the 24/4 and 27/7 COC regimens were not found to be equivalent in terms of 2- and 6-h SPMS. Equivalence was defined a priori as a mean within-patient

Fig. 1. Flowchart.

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1 (0–3) 2 (0–5) 0 (0–0) 0 (0–0)

1 (0–2) 4 (3–5) 0.5 (0–5) 0 (0–2)

.92 .1 .008 .008

treatment difference in SPMS measured on the last hormone-free day of each regimen such that the 95% CI of the mean difference did not cross − 1 or + 1 unit. The mean difference in SPMSs between the 24/4 and 21/7 regimens was 2.1 units (95% CI 0.7–3.5) at 2 h and 1.7 units (95% CI 0.4–3.0) at 6 h. The median difference was 0.5 unit (distribution free 95% CI 0–4) at 2 h and 0 unit (distribution free 95% CI 0–2) at 6 h. The median 2- and 6-h SPMSs on the last hormone-free day of the 24/4 and 21/7 regimens are shown in Fig. 2. There were significant differences in SPMSs at 2 and 6 h between mucus collected on the 24/4 and 21/7 regimens (p=.01 for both). On the 24/4 regimen, 3/18 individuals had good or excellent sperm penetrability (SPMS ≥ 7) at 2 h, and 2/18 had good or excellent sperm penetrability at 6 h. No subject had good or excellent sperm penetrability at 2 or 6 h while receiving the 21/7 regimen. The differences between the proportion of subjects having good or excellent SPMS on the 21/7 and 24/4 regimen were not statistically significant at 2 or 6 h (p=.25 and 0.5, respectively).

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This study demonstrated, as shown by extremely poor midcycle CMQ (mean CMQ=1) in both groups, that thickening of cervical mucus contributes to the efficacy of COCs. Because by study design subjects were not randomly sampled but had to achieve a CMQ of ≥ 10 to be enrolled, parametric statistical analysis of difference between the subjects’ own pretreatment mucus and their mucus on study drug cannot be determined. However, a mean CMQ of 1 is statistically different than a mean CMQ of 10 and indicates clinically impenetrable mucus. We also sought to examine the difference between a 24/4 and 21/7 regimen on CMQ and sperm penetration. We found no difference in CMQ between 24/4 and 21/7 on the last hormone-free day. However, the two regimens were not equivalent in terms of the SPMS, with subjects using the 24/4 regimen having more penetrable sperm at 2 and 6 h than the subjects using the 21/7 regimen. This finding could indicate a possible carryover effect or could be attributable to chance alone secondary to the relatively few number of subjects. Another hypothesis that we examined was that the presence of blood could enhance sperm penetration. More subjects had blood in their mucus on the 28th day when receiving the 24/4 regimen (16/18 on the last hormone-

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Sperm penetration meter score

4. Discussion

6

p=.01

p=.01

5 4 3 2 1

24/4

0 Difference

14 CMQ, median (IQR) 28 CMQ, median (IQR) 28 2hr SPMS, median (IQR) 28 6hr SPMS, median (IQR)

21/7

Day Day Day Day

24/4

21/7 regimen 24/4 regimen p Value

free day for the 24/4 regimen versus 8/18 on the last hormone-free day for the 21/7 regimen). However, there was no statistical trend towards greater sperm permeability for mucus containing blood among those subjects on the 21/7 regimen. Whether there was an effect from having blood in the mucus while on the 24/4 regimen is impossible to determine because all but two subjects on this regimen had blood in their mucus on day 28. The hypothesis that the presence of blood makes mucus more penetrable to sperm was not supported by the data. One limitation to this study is that we did not include ultrasound examination at each visit to evaluate follicular development and potential ovulation. Though studies have demonstrated that between 1.5% and 16.8% of women taking COCs ovulate [1–8], our study does not include ovarian follicular measurement either midcycle or on the last day of the pill-free interval. One area for future studies would be to examine the thickening of cervical mucus in women on COCs with documented follicular development. One strength of our study is the crossover design. Because the two COCs being studied both contain 20 mcg of ethinyl estradiol and 1 mg norethindrone acetate, study subjects should not have experienced any change in symptoms when being randomized to one arm from another. Because of the identical hormonal configuration, we were able to cross over the subjects without a washout period, which would have put

Difference

Table 2 Comparison of CMQ and SPMS

21/7

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Fig. 2. Median 2- and 6-h SPMSs on the final hormone-free day of 24/4 and 21/7 combined oral contraception regimens (n=18).

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all of our subjects at risk of unintended pregnancy. The crossover design allows us to use each subject as her own control, essentially eliminating bias secondary to individual confounders. A period-treatment trend suggested that taking the 21/7 regimen first contributed to greater mucus permeability on the 24/4 regimen in the fourth month; however, we could find no clinical correlation to this statistical finding. This study was powered as an equivalency trial because we did not know a priori that the two regimens would be different in terms of quality or sperm penetration. Therefore, the study was not powered to detect whether one regimen was statistically significantly superior to the other. In summary, the clinical significance of a half-a-point difference in SPMS is likely negligible. While this is interesting, is difficult to interpret and may even warrant further investigation, it likely has little clinical relevance because all subjects showed poor quality and impenetrable mucus on the last day of the hormone-free interval. Though Dinger et al. [21] showed increased efficacy of 24/4 compared with 21/7 in a large observation study, the results of our study suggest that cervical mucus changes associated with the 24/4 regimen may not contribute to greater contraceptive efficacy. Although evidence suggests that cervical mucus changes are a primary mechanism of action of progestin-only contraceptives, no study has demonstrated the efficacy of cervical mucus as a barrier to sperm penetration in users of COCs. Our study confirms that even after 7 hormone-free days, the mucus of COC users remains of poor quality and impenetrable to sperm. There are many misperceptions regarding the mechanism of action of COCs that contribute to low use in certain areas of the United States, including the misperception that they act as abortifacients. This study clarifies that COCs alter the cervical mucus so as to inhibit sperm transport, thereby preventing fertilization even in the presence of ovulation. References [1] Coney P, Del Conte A. The effects on ovarian activity of a monophasic oral contraceptive with 100 μg levonorgestrel and 20 μg ethinyl estradiol. Am J Obstet Gynecol 1999;181:S53–8. [2] Pierson RA, Archer DF, Moreau M, Shangold GA, Fisher AC, Creasy GW. Ortho Evra™/Evra™ versus oral contraceptives: follicular development and ovulation in normal cycles and after an intentional dosing error. Ortho Evra™/Evra™ 008 Study Group. Fertil Steril 2003;80:34–42. [3] Spona J, Elstien M, Feichtinger W, et al. Shorter pill-free interval in combined oral contraceptives decreases follicular development. Contraception 1996;54:71–7. [4] van der Does J, Exalto N, Dieben T, Coelingh Bennink H. Ovarian activity suppression by two different low-dose triphasic oral contraceptives. Contraception 1995;52:357–61. [5] Grimes DA, Godwin AJ, Rubin A, Smith JA, Lacarra M. Ovulation and follicular development associated with three low-dose oral contraceptives: a randomized controlled trial. Obstet Gynecol 1994;83:29–34. [6] Hamilton CJ, Hoogland HJ. Longitudinal ultrasonographic study of the ovarian suppressive activity of a low-dose triphasic oral contraceptive during correct and incorrect pill intake. Am J Obstet Gynecol 1989;161:1159–62.

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