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Effect on ovarian activity and ovulation inhibition of different oral dosages of levonorgestrel

      Abstract

      Objectives

      To investigate the effect of different oral dosages of levonorgestrel (LNG) on ovarian activity and to identify the lowest dosage at which no ovulation occurred. Secondary objectives were to assess return of ovulation after stopping treatment, bleeding pattern, pharmacokinetic (PK) parameters and safety and tolerability.

      Study design

      A parallel-group study with adaptive design was performed in 90 healthy women with proven ovulatory cycles. Investigated dosages were LNG 0.095, 0.115 and 0.135 mg per day. Measurements of follicular growth and estradiol (E2) and progesterone concentrations were performed every 3 (±1) days during a 56-day treatment and a post-treatment period. Follicle-stimulating hormone and luteinizing hormone concentrations and multiple-dose PK parameters were determined during treatment.

      Results

      Two normal ovulations occurred in the LNG 0.095 mg group, none in the higher dose groups. Most subjects had active follicle-like structures without ovulation (Hoogland-Skouby scores 4). Ovarian activity was more suppressed in the highest dose group than in the other groups. Mean E2 concentrations were 241, 219 and 180 pmol/L during treatment with 0.095, 0.115 and 0.135 mg per day, respectively. PK results showed dose-proportionality. Most subjects ovulated during the post-treatment period.

      Conclusion

      LNG 0.115 mg per day was the lowest effective dosage for consistent ovulation inhibition. All investigated dosages were safe and well-tolerated, and mean E2 concentrations were sufficient for prevention of hypoestrogenic side effects.

      Implications

      Marketed progestogen-only pills (POP) containing 0.03 mg LNG do not consistently inhibit ovulation. Increasing the dosage to 0.115 mg or 0.135 mg per day, resulting in consistent ovulation inhibition, may improve the contraceptive efficacy of the LNG-POP.

      Keywords

      1. Introduction

      Levonorgestrel (LNG) is a well-known synthetic progestogen that has been used in hormonal contraceptive preparations for decades. In the 1970s LNG was marketed as a low-dose progestogen-only pill (POP), in a dosage of 0.030 mg per day [
      • McCann MF
      • Potter LS.
      Progestin-only oral contraception: a comprehensive review.
      ]. POPs are still used, in particular by women with contraindications for the estrogen component in combined oral contraceptives (COCs), such as women with risk factors for cardiovascular diseases [
      World Health Organization
      Medical eligibility criteria for contraceptive use: a WHO family planning cornerstone.
      ].
      The contraceptive efficacy of POPs is based on several mechanisms of action [
      • McCann MF
      • Potter LS.
      Progestin-only oral contraception: a comprehensive review.
      ]. Progestogens decrease cervical mucus permeability, fallopian tube motility, and endometrial receptivity. The pituitary-ovarian function is affected: follicular growth, the midcycle gonadotropin peak, ovulation, and/or luteal function may be disturbed or suppressed [
      • Moghissi KS.
      Morphologic changes in the ovaries of women treated with continuous microdose progestogens.
      ,
      • Moghissi KS
      • Syner FN
      • McBride L.
      Contraceptive mechanism of microdose norethindrone.
      ,
      • Landgren B-M
      • Balogh A
      • Shin MW
      • Lindberg M
      • Diczfalusy E.
      Hormonal effects of the 300 µg` norethisterone (NET) minipill. 2. Daily gonadotropin levels in 43 subjects during a pretreatment cycle and during the second month of NET administration.
      ]. The effect on pituitary-ovarian function is dose-dependent and more pronounced with higher progestogen dosages [
      • Spona J
      • Schneider WHF
      • Ch Bieglmayer
      • Schroeder R
      • Pirker R.
      Ovulation inhibition with different doses of levonorgestrel and other progestogens: clinical and experimental investigations.
      • Klipping C
      • Duijkers I
      • Remmers A
      • Faustmann T
      • Zurth C
      • Klein S
      • et al.
      Ovulation-inhibiting effects of dienogest in a randomized, dose-controlled pharmacodynamic trial of healthy women.
      ].
      The dosage of a POP containing desogestrel 0.075 mg is sufficiently high to inhibit ovulation in at least 98% of cycles, compared with 72% of cycles with LNG 0.030 mg [
      • Rice C
      • Killick S
      • Hickling D
      • Coelingh Bennink H
      Ovarian activity and vaginal bleeding patterns with a desogestrel-only preparation at three different doses.
      ,
      • Rice CF
      • Killick SR
      • Dieben T
      • Coelingh Bennink H
      A comparison of the inhibition of ovulation achieved by desogestrel 75 µg and levonorgestrel 30 µg daily.
      ,
      • Duijkers IJM
      • Heger-Mahn D
      • Drouin D
      • Skouby S.
      A randomised study comparing the effect on ovarian activity of a progestogen-only pill (POP) containing desogestrel and a new POP containing drospirenone in a 24/4 regimen.
      ]. Probably due to consistent ovulation inhibition, which is considered the most robust mechanism of action of a POP, the contraceptive efficacy of desogestrel 0.075 mg is higher than that of LNG 0.030 mg and other low-dose POPs, and comparable to the efficacy of COCs [
      • McCann MF
      • Potter LS.
      Progestin-only oral contraception: a comprehensive review.
      ,
      Collaborative Study Group on the Desogestrel-containing Progestogen-only Pill
      A double-blind study comparing the contraceptive efficacy, acceptability and safety of two progestogen-only pills containing desogestrel 75 µg/day or levonorgestrel 30 µg/day.
      ].
      Increasing the dosage of the LNG-POP, resulting in consistent inhibition of ovulation, is therefore expected to improve its contraceptive efficacy. Furthermore, previous studies with non-oral LNG preparations suggested that increasing the LNG dosage might reduce bleeding irregularities, a common reason for women to discontinue POP use [
      • Mishell DR
      • Lumkin M.
      Initial clinical studies of intravaginal rings containing norethindrone and norgestrel.
      ,
      • Faundes A
      • Brache de Mejias V
      • Leon P
      • Robertson D
      • Alvarez F.
      First year clinical experience with six levonorgestrel rods as subdermal contraception.
      ]. We, therefore, decided to develop a new higher-dosed LNG-POP, aiming for an estrogen-free contraceptive with high contraceptive reliability and an acceptable bleeding pattern.
      As to our knowledge, the oral LNG dosage that is required for consistent ovulation inhibition is not known yet. We are only aware of one previous study in 12 women, investigating serum hormone levels during oral administration of LNG 0.030, 0.050, 0.100, and 0.150 mg for 21 days [
      • Spona J
      • Schneider WHF
      • Ch Bieglmayer
      • Schroeder R
      • Pirker R.
      Ovulation inhibition with different doses of levonorgestrel and other progestogens: clinical and experimental investigations.
      ]. The ovulation-inhibitory dose was found to be 0.050 mg per day, however, this conclusion was based on only 2 to 4 women per dose group. Our group previously performed a proof-of-concept study in 20 women, treated with LNG 0.060 or 0.090 mg per day during 56 days [
      • Oettel M
      • Kochhar PS
      • Osterwald H
      • Zarapkar MS
      • Walawalkar R.
      The Progestin-Only Pill (POP) is not a niche option: new preclinical and clinical data about the interrelations between levonorgestrel-dose, peripheral as well as central contraceptive effects and bleeding behaviour.
      ]. Progesterone (P) concentrations were measured on treatment days 24 and 48. Ovulatory P levels were detected in 3 of 10 women in the 0.060 mg group and in none of the 10 women in the 0.090 mg group, so the ovulation-inhibitory dose appeared to be higher than 0.060 mg per day.
      The present dose-finding study in healthy premenopausal women further investigated the effect of different dosages of LNG, administered orally for 56 days, on ovarian activity to identify the lowest dose at which no ovulation occurred. Secondary aims of the study were to assess the return of ovulation after stopping treatment, bleeding pattern, pharmacokinetic (PK) parameters, and safety and tolerability of the different dosages.

      2. Material and methods

      We conducted this single-center, open-label, parallel-group, adaptive-design study in a clinical research center (dinox GmbH, Berlin, Germany) in 2019, adhering to the principles of Good Clinical Practice and the Declaration of Helsinki. An independent ethics committee (Landesamt für Gesundheit und Soziales, Berlin, Germany) approved the study protocol and all subjects provided written informed consent.

      2.1 Subjects

      Study participants were healthy female volunteers, aged 18−45 years, having a body mass index (BMI) ≥18 kg/m2, non-smoking if aged >30 years or smoking maximally 10 cigarettes per day if aged ≤30 years. The main exclusion criteria were: liver diseases, risk factors for venous or arterial thromboembolic diseases, known cycle irregularities, pregnancy or lactation in the last 3 cycles, and diseases or use of medication that might affect safety, absorption, or PK of the study medication. The subjects had to use barrier contraceptives or refrain from heterosexual activity during the study.

      2.2 Treatment

      The study had an adaptive design. We planned maximally five treatment groups, each consisting of 30 women, at least 5 of which had a BMI of ≥30 kg/m2 at screening. Planned dosages were 0.060 mg, 0.075 mg, 0.095 mg, 0.115 mg or 0.135 mg LNG per day (Naari Pharma Private Limited, India).
      The first tested dosage was 0.095 mg per day. The choice of subsequent dosages depended on the occurrence of ovulations in the previous treatment groups. The study was stopped when the lowest dosage completely inhibiting ovulation was identified, and one higher and one lower dosage was investigated.

      2.3 Assessments

      2.3.1 Ovarian activity

      After a screening examination, subjects had a washout cycle if they used hormonal contraceptives. A pre-treatment cycle was monitored to assess if the subjects had ovulatory cycles. We measured follicular growth by transvaginal ultrasonography (TVUS) every three (±1) days from cycle day 9 (±1) onwards. If TVUS showed follicle rupture, P concentrations were determined 2 (±1) and, if necessary, 4 (±1) days thereafter. The subject was eligible if ovulation occurred before or on day 27 (±1) and the P concentration 2 or 4 days after follicle rupture was ≥16 nmol/L.
      Subjects started study medication intake on the first day they woke up with menstrual bleeding after the pre-treatment cycle. One tablet was taken orally, around the same time every morning, for 56 consecutive days. Study visits were planned every 3 (±1) days from treatment day 3 (±1) until 27 (±1) and from day 31 (±1) until day 55 (±1). At each visit the diameter of the largest follicle-like structure (FLS) and serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and P concentrations were measured.
      Subjects were excluded from the study if an active FLS was present on treatment day 3 (±1). If ovulation was suspected by TVUS, additional visits for P determinations were planned 2, 4, and 6 days after suspected ovulation.
      After the last treatment day, assessments were continued every 3 (±1) days until ovulation was observed, followed by P measurements 2 (±1) and, if necessary, 4 (±1) days after ovulation. An end-of-study examination was performed 6 (±1) days after ovulation, or on day 30 (±1) in case no ovulation had been observed until then.

      2.3.2 Sex hormone-binding globulin (SHBG) and PK

      Serum SHBG concentrations were determined after ovulation in the pre-treatment cycle, on treatment day 55 (±1), and at the end-of-study examination. Multiple-dose LNG PK parameters were investigated in approximately 15 subjects per dose group, 5 of them having a BMI ≥30 kg/m2. Blood samples were taken on treatment day 55 (±1) pre-dose and 0.5, 1, 2, 3, 4, 6 and 24 hours after dosing.

      2.3.3 Vaginal bleeding

      Throughout the study, subjects kept a daily record of vaginal bleeding. During the treatment period, they also documented the time of study medication intake.

      2.3.4 Safety

      At every visit the subjects were questioned for adverse events and use of concomitant medication. Safety laboratory and pregnancy tests were performed at regular intervals. Physical examinations were performed at screening and end of study.

      2.4 Analysis

      The primary outcome parameter was ovarian activity according to the Hoogland-Skouby score (Table 1), determined for each 28-day treatment period [
      • Hoogland HJ
      • Skouby SO.
      Ultrasound evaluation of ovarian activity under oral contraceptives.
      ]. If a luteinized unruptured follicle or ovulation was observed during treatment (Hoogland-Skouby score 5 or 6), the subsequent luteal phase was evaluated using the Landgren criterion, i.e., the luteal phase was considered normal when P concentrations were >16 nmol/L during at least 5 days [
      • Landgren B-M
      • Diczfalusy E.
      Hormonal effects of the 300 µg norethisterone (NET) minipill. 1. Daily steroid levels in 43 subjects during a pretreatment cycle and during the second month of NET administration.
      ]. TVUS measurements of the diameter of the largest FLS (mean of two directions) were performed using a Voluson E8 Expert device (GE Medical Systems). Serum FSH, LH, E2, P, and SHBG concentrations were determined by chemiluminescent microparticle immunoassays (Abbott, Longford, Ireland, see Appendix A for additional details). All pharmacodynamic parameters were evaluated descriptively.
      Table 1Hoogland-Skouby score distribution during two 28-day treatment periods with oral levonorgestrel (LNG) 0.095 mg, 0.115 mg or 0.135 mg per day
      Hoogland-Skouby scoreFLS diameter (mm)LNG 0.095 mgLNG 0.115 mgLNG 0.135 mg
      E2 (pmol/L)P (nmol/L)Treatment period 1Treatment period 2Treatment period 1Treatment period 2Treatment period 1Treatment period 2
      n%n%n%n%n%n%
      1No activity≤10--13.70013.713.8514.014.0
      2Potential activity10 < FLS ≤13--829.6624.0622.2623.1832.0624.0
      3Non-active FLS>13≤100-13.70013.727.70014.0
      4Active FLS>13>100≤51555.61976.01970.41765.41664.01768.0
      5LUF>13, persistent>100>5000000000000
      6‘Ovulation’>13, ruptured>100>527.40000000000
      N total272527262525
      FLS, follicle-like structure; LUF, luteinized unruptured follicle.
      Adapted from Hoogland and Skouby, 1993
      • Hoogland HJ
      • Skouby SO.
      Ultrasound evaluation of ovarian activity under oral contraceptives.
      .
      Plasma LNG concentrations were determined by a validated liquid chromatography with tandem mass spectrometry (LCMS-MS) method (ACC, Leidersbach, Germany, see Appendix A). PK parameters were derived by means of non-compartmental analysis. Descriptive statistics per treatment group and subgroup (BMI <30 and ≥30 kg/m2) as well as regression analysis after dose adjustment were performed. In addition, we evaluated the frequency of LNG concentrations ≤180 pg/mL, a previously defined efficacy threshold level in LNG implant users [
      • Sivin I
      • Wan L
      • Ranta S
      • Alvarez F
      • Brache V
      • Mishell DR
      • et al.
      Levonorgestrel concentrations during 7 years of continuous use of Jadelle contraceptive implants.
      • Westhoff CL
      • Chen BA
      • Jensen JT
      • Barnhart K
      • Thomas M
      • Teal S
      • et al.
      Phase I/II pharmacokinetic and pharmacodynamic evaluation of two levonorgestrel-only contraceptive patches in a multicenter randomized trial.
      ].

      3. Results

      3.1 Treatment groups

      Figure 1 shows the subject disposition. The mean (±standard deviation (SD)) age was 33.3 (±6.2) years, mean BMI was 25.4 (±4.9) kg/m2 in the entire group, 23.7 (±3.0) kg/m2 in the low and 33.5 (±3.1) kg/m2 in the high-BMI subgroup.
      Fig 1
      Fig. 1Disposition of subjects in a dose-finding study of oral levonorgestrel for contraception.
      We investigated three dosages: LNG 0.095 mg, 0.115 mg, and 0.135 mg. In accordance with the stop criterion for this adaptive-design study, we did not investigate the two lower dosages (0.060 and 0.075 mg). All women participated in only one dose group.

      3.2 Ovarian activity

      Table 1 shows the distribution of Hoogland-Skouby scores. Two subjects (BMI 30.8 and 22.0 kg/m2) in the LNG 0.095 mg group ovulated in treatment period 1, with adequate luteal phases according to the Landgren criterion (P >16 nmol/L during ≥5 days). The lowest dosage at which no ovulation occurred was LNG 0.115 mg per day. Also in the highest dose group (0.135 mg) none of the subjects ovulated as defined by the Hoogland-Skouby score. One subject in this group (BMI 20.4 kg/m2) had an FLS of 12.3 mm, E2 concentration of 378 pmol/l and LH concentration of 7.79 IU/L on treatment day 3. The FLS was 7.1 mm on day 5, and P concentrations were between 6.64 and 12.19 nmol/L from days 5 to 11 (Hoogland-Skouby score 2).
      In all treatment groups the majority of subjects had Hoogland-Skouby score 4, i.e., active FLS. Except for the 2 ovulations in the lowest dose group, there was no clear tendency towards lower scores, i.e., more ovarian suppression, with increasing LNG doses.
      Table 2 shows the maximum diameter of the largest FLS and maximum and mean E2 concentrations per subject per cycle and over the entire treatment period. Figure 2 depicts the courses of mean FLS diameter and hormone concentrations over time. The results are in agreement with the Hoogland-Skouby scores, in most subjects follicular growth with corresponding increases of E2 concentrations occurred during treatment. FLS diameters and E2 concentrations were comparable in the 0.095 and 0.115 mg groups. In the 0.135 mg group, FLS diameter was smaller in both treatment cycles and E2 concentrations were lower in the first treatment period, indicating that more suppression of follicular growth occurred in the highest dose group. Differences between the groups were smaller in treatment period 2, E2 concentrations being stable and lower than in period 1. Mean values of average E2 concentrations per subject over the entire treatment period were 241, 219, and 180 pmol/L in the 0.095, 0.115, and 0.135 mg groups, respectively. With all dosages, mean E2 concentrations were ≥110 pmol/L (30 pg/mL) in 80 to 90% of subjects and ≥73.4 pmol/L (20 pg/mL) in all subjects, except in one subject in the 0.115 mg group (low-BMI subgroup, 50 pmol/L, 13.6 pg/mL).
      Table 2Descriptive statistics of maximum diameters of the largest follicle-like structure (FLS), maximum estradiol (E2) concentrations and mean E2 concentrations per subject during two 28-day treatment periods with oral levonorgestrel (LNG) 0.095 mg, 0.115 mg or 0.135 mg per day, in the entire treatment group and in subgroups of subjects with body mass index (BMI) <30 and ≥30 kg/m2
      LNG 0.095 mgLNG 0.115 mgLNG 0.135 mg
      MaximumFLS diameter (mm)Treatment period 1N262725
      Mean ± SD20.0 ± 8.620.4 ± 7.916.6 ± 5.7
      Median (range)20.6 (9.7 – 37.3)20.3 (7.3 – 36.0)13.9 (7.3 – 28.4)
      Treatment period 2N252625
      Mean ± SD20.5 ± 7.519.9 ± 7.517.4 ± 5.0
      Median (range)20.2 (10.5 – 34.1)20.8 (9.9 – 35.8)16.8 (8.8 – 27.8)
      Entire treatment period
      All subjectsN252625
      Mean ± SD22.1 ± 7.921.4 ± 7.718.5 ± 5.3
      Median (range)22.5 (10.5 – 37.3)20.8 (10.4 – 36.0)18.1 (8.8 – 28.4)
      BMI <30 kg/m2N202120
      Mean ± SD22.5 ± 8.219.8 ± 7.518.4 ± 5.0
      Median (range)22.4 (10.5 – 37.3)19.2 (10.4 – 36.0)18.2 (8.8 – 27.8)
      BMI ≥30 kg/m2N555
      Mean ± SD20.6 ± 6.927.9 ± 4.419.0 ± 7.1
      Median (range)22.5 (11.8 – 27.8)27.2 (22.0 – 34.3)15.3 (13.0 – 28.4)
      Maximum E2 concentration (pmol/L)Treatment period 1N262725
      Mean ± SD595 ± 495563 ± 551369 ± 245
      Median (range)362 (99 – 1820)481 (110 – 2270)239 (110 – 892)
      Treatment period 2N252625
      Mean ± SD384 ± 304367 ± 278302 ± 184
      Median (range)224 (110 – 1130)215 (103 – 995)242 (128 – 811)
      Entire treatment period
      All subjectsN252625
      Mean ± SD660 ± 500610 ± 559416 ± 256
      Median (range)580 (110 – 1820)511 (128 – 2270)279 (128 – 892)
      BMI <30 kg/m2N202120
      Mean ± SD705 ± 537611 ± 623415 ± 244
      Median (range)550 (110 – 1820)410 (130 – 2270)340 (150 – 890)
      BMI ≥30 kg/m2N555
      Mean ± SD484 ± 277607 ± 116421 ± 332
      Median (range)580 (150 – 840)540 (540 – 810)240 (130 – 820)
      Mean E2 concentration (pmol/L)Treatment period 1N262725
      Mean ± SD289 ± 195269 ± 217198 ± 100
      Median (range)231 (74 – 761)244 (45 – 921)156 (84 – 390)
      Treatment period 2N252625
      Mean ± SD186 ± 97170 ± 88162 ± 62
      Median (range)144 (72 – 391)146 (47 – 354)148 (67 – 310)
      Entire treatment period
      All subjectsN252625
      Mean ± SD241 ± 130219 ± 146180 ± 73
      Median (range)204 (81 – 472)186 (46 – 631)144 (77 – 311)
      BMI <30 kg/m2N202120
      Mean ± SD253 ± 140213 ± 160178 ± 72
      Median (range)220 (80 – 470)160 (50 – 630)160 (80 – 310)
      BMI ≥30 kg/m2N555
      Mean ± SD190 ± 72242 ± 66190 ± 87
      Median (range)200 (110 – 260)210 (180 – 340)140 (100 – 310)
      SD, standard deviation.
      Fig 2
      Fig. 2Mean follicular diameters, estradiol (E2), progesterone (P), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) serum concentrations over time during and after 56 days of oral treatment with levonorgestrel (LNG) 0.095 mg, 0.115 mg or 0.135 mg per day.
      Mean FSH concentrations slightly decreased during treatment period 1, due to negative feedback by increasing E2 levels. Individual LH concentrations were all below 12.1 IU/L, except for an LH peak of 68.0 IU/L in one of the ovulating subjects in the 0.095 mg group. In the other ovulating subject no pre-ovulatory LH peak was observed. All individual P concentrations were below 5 nmol/L during treatment, except in the 2 ovulating subjects in the 0.095 mg group, the subject in the 0.135 mg group described above, and another subject with a single P concentration of 5.85 nmol/L at the first treatment visit, probably a residue from the pre-treatment cycle.
      Comparison of maximum FLS diameters and E2 concentrations in the two BMI groups did not show clear differences (Table 2).

      3.3 Return of ovulation

      In the majority of subjects ovulation occurred within the 30-day post-treatment period, followed by P concentrations ≥16 nmol/L. In 1 or 2 subjects per dose group P concentrations after ovulation remained below 16 nmol/L (4 subjects with low BMI and one with high BMI). In 1 or 2 subjects per group ovulation did not occur within 30 days (3 with low and 2 with high BMI). Ovulation was observed on average 15.5, 14.1, and 12.8 days after last treatment in the 0.095, 0.115, and 0.135 mg groups, respectively.

      3.4 SHBG

      In all treatment groups, SHBG concentrations were suppressed by approximately 60% at the end of treatment period 2 (Table 3). At the end-of-study examination, on average 22 days after the last treatment, mean SHBG concentrations had not yet returned to baseline values. At all timepoints, mean SHBG concentrations were lower in the subgroups with BMI ≥30 kg/m2 than in the low-BMI subgroups.
      Table 3Descriptive statistics of sex hormone-binding globulin (SHBG) concentrations (nmol/L) before, during and after 56 treatment days with oral levonorgestrel (LNG) 0.095 mg, 0.115 mg or 0.135 mg per day. SHBG concentrations in the pre-treatment cycle, at day 55 ±1 of treatment and at the end-of-study examination are listed for the entire treatment group and for subgroups of subjects with body mass index (BMI) <30 and ≥30 kg/m2
      LNG 0.095 mgLNG 0.115 mgLNG 0.135 mg
      SHBG (nmol/L)Pre-treatment
      All subjectsN262725
      Mean ± SD66.0 ± 26.465.4 ± 30.366.8 ± 25.8
      Median (range)63.4 (19.8 – 132.3)68.9 (22.1 – 155.9)61.9 (24.3 – 122.5)
      BMI <30 kg/m2N212220
      Mean ± SD72.9 ± 24.271.9 ± 28.773.2 ± 23.6
      Median (range)69.1 (29.1 – 132.3)74.7 (29.9 – 155.9)71.9 (35.6 – 122.5)
      BMI ≥30 kg/m2N555
      Mean ± SD37.3 ± 12.636.9 ± 20.441.1 ± 17.6
      Median (range)37.1 (19.8 – 51.8)32.5 (22.1 – 72.4)39.6 (24.3 – 65.0)
      Treatment day 55 ±1
      All subjectsN262625
      Mean ± SD27.7 ± 10.623.9 ± 12.124.0 ± 9.5
      Median (range)25.5 (9.4 – 50.2)18.9 (8.7 – 56.3)23.7 (10.1 – 49.2)
      BMI <30 kg/m2N212120
      Mean ± SD30.3 ± 9.926.6 ± 11.926.1 ± 9.1
      Median (range)27.4 (12.0 – 50.2)21.6 (12.3 – 56.3)24.7 (12.2 – 49.2)
      BMI ≥30 kg/m2N555
      Mean ± SD17.0 ± 6.212.6 ± 3.415.6 ± 5.9
      Median (range)19.1 (9.4 – 24.0)11.2 (8.7 – 16.8)13.6 (10.1 – 23.6)
      End of study
      All subjectsN262725
      Mean ± SD49.9 ± 17.448.1 ± 20.158.6 ± 28.5
      Median (range)47.7 (18.1 – 80.9)46.7 (16.7 – 99.4)47.8 (21.7 – 128.3)
      BMI <30 kg/m2N212220
      Mean ± SD54.5 ± 15.353.5 ± 17.764.8 ± 28.2
      Median (range)52.9 (20.6 – 80.9)50.5 (28.0 – 99.4)52.5 (31.6 – 128.3)
      BMI ≥30 kg/m2N555
      Mean ± SD30.5 ± 11.524.7 ± 12.433.9 ± 11.1
      Median (range)31.8 (18.1 – 44.9)19.5 (16.7 – 46.7)35.0 (21.7 – 47.8)
      SD, standard deviation.

      3.5 PK

      Figure 3A shows mean LNG concentrations on day 55 ±1. Maximum values were reached 1 hour after dosing, with a rapid decrease thereafter. Descriptive statistics of PK parameters are listed in Table 4, showing increased AUC0-24,ss, Cmax,ss, Cav and Cmin,ss values with increasing dose. Geometric means of Cmax,ss, Cav and Cmin,ss were higher than 180 pg/mL in all dose groups, as well as all individual Cmax,ss and Cav values. Some women in the 0.095 and 0.115 mg groups had Cmin,ss values below 180 pg/mL, whereas all individual Cmin.ss levels in the 0.135 mg group were higher than 180 pg/mL.
      Fig 3
      Fig. 3Mean plasma levonorgestrel (LNG) concentrations after 55 (±1) days of oral treatment with LNG 0.095 mg, 0.115 mg or 0.135 mg per day, in the entire study group (A: 17, 17 and 16 subjects per dose group, respectively), in the subgroups with BMI <30 kg/m2 (B: 12, 12 and 11 subjects, respectively) and in the subgroups with BMI ≥30 kg/m2 (C: 5 subjects per dose group).
      Table 4Descriptive statistics of pharmacokinetic (PK) parameters after oral multiple-dose administration of 0.095 mg, 0.115 mg or 0.135 mg levonorgestrel (LNG) per day, respectively, over 55 ±1 consecutive days, in the entire treatment group and in subgroups with body mass index (BMI) <30 and ≥30 kg/m2, and numbers of subjects per group with maximum, minimum and average LNG concentrations below 180 pg/mL
      LNG 0.095 mgLNG 0.115 mgLNG 0.135 mg
      ParameterAUC0-24,ss (h*pg/mL)Cmax,ss (pg/mL)Cmin,ss (pg/mL)Cav (pg/mL)CLss (L/h)AUC0-24,ss (h*pg/mL)Cmax,ss (pg/mL)Cmin,ss (pg/mL)Cav (pg/mL)CLss (L/h)AUC0-24,ss (h*pg/mL)Cmax,ss (pg/mL)Cmin,ss (pg/mL)Cav (pg/mL)CLss (L/h)
      All subjects
      N171717171717171717171616161616
      Mean1310016502865488.141480017903136188.991750020104067288.32
      SD490060399.32042.7454806361392284.0250706041542112.31
      Min72909031613044.0955807301142334.401020011401894234.51
      Median1180016302864918.061290018102985378.921690019303657038.01
      Max23200312049096713.0261002830591109020.6300003230699125013.3
      Geometric Mean1240015502715157.691380016702815768.311680019203807018.02
      CV% Geometric Mean36.9237.3835.3136.9236.9241.2841.0252.6341.2841.2828.7430.8539.2328.7428.74
      N < 180 pg/mL030050000
      N ≥ 180 pg/mL171417171217161616
      BMI <30 kg/m2
      N121212121212121212121111111111
      Geometric mean1400018302935826.801630020303086807.051760021403837337.68
      CV% Geometric mean34.1527.7137.0134.1534.1529.4725.1153.0229.4729.4724.6026.6730.2224.6024.60
      N < 180 pg/mL020030000
      N ≥ 180 pg/mL12101212912111111
      BMI ≥30 kg/m2
      N555555555555555
      Geometric mean9200105022438310.39320105022738812.31530015203736378.83
      CV% Geometric mean23.4523.6223.5723.4523.4536.3526.6649.1536.3536.3537.8426.8261.1537.8437.84
      N < 180 pg/mL010020000
      N ≥ 180 pg/mL545535555
      AUC0-24,ss, area under the serum concentration vs time profile from dosing at treatment day 55 ±1 to 24 hours after administration (dosing interval τ); Cmax,ss, maximum concentration within the dosing interval τ; Cmin,ss, (absolute) minimum concentrations within the dosing interval τ, obtained directly from measured values; Cav, average concentration at steady-state, Cav = AUC0-τ/τ; CLss, apparent total body clearance, dose / AUC.
      Regression analysis showed dose proportionality for total exposure and a trend to negligibly lower maximum exposure with increasing doses (Supplemental Figure 1). For all dosages a trend to lower exposure, lower Cmax,ss and higher clearance was observed in the subgroups with BMI ≥30 kg/m2 (Table 4, Figs. 3B and C).

      3.6 Vaginal bleeding

      The mean (±SD) number of days with bleeding or spotting during the 56-day treatment period, excluding the first seven treatment days, was 12.3 (±8.6), 14.5 (±9.4) and 16.0 (±11.5) in the 0.095, 0.115 and 0.135 mg groups, respectively, median values were 11.5, 14.5 and 13.0 days (Supplemental Table 1). Spotting occurred most frequently, if bleeding occurred, it was mainly light bleeding.

      3.7 Safety

      The study medication was well tolerated. Most frequently reported adverse events that were assessed by the investigator as related to the study medication were headache, altered mood, acne, decreased SHBG concentrations, and ovarian cysts (Supplemental Table 2). One subject discontinued prematurely because of dizziness, that was possibly related to study treatment. One serious adverse event occurred (pneumonia), that was not related to treatment. There were no clinically relevant changes in physical examination or routine laboratory results. One subject became pregnant during the follow-up period, conception occurring well after the last study treatment.

      4. Discussion

      The study results showed that increasing the dosage of the LNG-POP resulted in consistent inhibition of normal ovulations. The lowest effective dosage was 0.115 mg per day, which is higher than the previously assumed ovulation-inhibitory dose of 0.050 to 0.060 mg [
      • Spona J
      • Schneider WHF
      • Ch Bieglmayer
      • Schroeder R
      • Pirker R.
      Ovulation inhibition with different doses of levonorgestrel and other progestogens: clinical and experimental investigations.
      ,
      • Schindler AE
      • Campagnoli C
      • Druckmann R
      • Huber J
      • Pasqualini JR
      • Schweppe KW
      • et al.
      Classification and pharmacology of progestins.
      • Taubert H-D
      • Kuhl H.
      Kontrazeption mit Hormonen, ein Leitfaden für die Praxis.
      ]. Although in most subjects follicular growth and concomitant increases of E2 concentrations occurred, LH surges and normal ovulations were inhibited during treatment with LNG 0.115 or 0.135 mg per day.
      One subject in the 0.135 mg group did not ovulate according to the Hoogland-Skouby score but may have had dysfunctional ovulation of a small follicle at the start of treatment, preceded by a blunted LH increase and followed by an inadequate luteal phase. Previous studies reported similar dysfunctional ovulations at the start of treatment, that seemed to be triggered by initiation of progestogen treatment in the presence of a growing follicle [
      • Brache V
      • Blumenthal PD
      • Alvarez F
      • Dunson TR
      • Cochon L
      • Faundes A.
      Timing of onset of contraceptive effectiveness in Norplant® implant users.
      ]. Also during the course of progestogen-only treatment abnormal ovulations with inadequate luteal phases regularly occur and are not expected to result in implantation and pregnancy [
      • McCann MF
      • Potter LS.
      Progestin-only oral contraception: a comprehensive review.
      ,
      • Landgren B-M
      • Diczfalusy E.
      Hormonal effects of the 300 µg norethisterone (NET) minipill. 1. Daily steroid levels in 43 subjects during a pretreatment cycle and during the second month of NET administration.
      ,
      • Brache V
      • Blumenthal PD
      • Alvarez F
      • Dunson TR
      • Cochon L
      • Faundes A.
      Timing of onset of contraceptive effectiveness in Norplant® implant users.
      ].
      We found that LNG 0.115 and 0.135 mg inhibited ovulation to the same extent as desogestrel 0.075 mg, a POP with contraceptive efficacy similar to combined pills [
      • Rice CF
      • Killick SR
      • Dieben T
      • Coelingh Bennink H
      A comparison of the inhibition of ovulation achieved by desogestrel 75 µg and levonorgestrel 30 µg daily.
      ,
      • Duijkers IJM
      • Heger-Mahn D
      • Drouin D
      • Skouby S.
      A randomised study comparing the effect on ovarian activity of a progestogen-only pill (POP) containing desogestrel and a new POP containing drospirenone in a 24/4 regimen.
      ]. The contraceptive efficacy of the higher-dosed LNG-POP will have to be confirmed in future studies.
      Progestogen-only preparations generally do not completely suppress follicular growth and E2 production [
      • McCann MF
      • Potter LS.
      Progestin-only oral contraception: a comprehensive review.
      ]. This prevents the occurrence of hypoestrogenic side effects, in particular bone loss. In all LNG treatment groups mean E2 concentrations well above 110 pmol/L (30 pg/mL), the lower limit assumed not to lead to accelerated bone loss [
      • Barbieri RL.
      Hormone treatment of endometriosis: the estrogen threshold hypothesis.
      ,
      • Riggs MM
      • Bennetts M
      • van der Graaf PH
      • Martin SW.
      Integrated pharmacometrics and systems pharmacology model-based analysis to guide GnRH receptor modulator development for management of endometriosis.
      ,
      • Hadji P
      • Colli E
      • Regidor P-A.
      Bone health in estrogen-free contraception.
      ].
      The PK results are in line with previous PK studies with orally administered LNG [
      • Kuhnz W
      • Al-Yacoub G
      • Fuhrmeister A.
      Pharmacokinetics of levonorgestrel in 12 women who received a single oral dose of 0.15 mg levonorgestrel and, after a washout phase, the same dose during one treatment cycle.
      • Hofmann BM
      • Apter D
      • Bitzer J
      • Reinecke I
      • Serrani M
      • Höchel J
      • et al.
      Comparative pharmacokinetic analysis of levonorgestrel-releasing intrauterine systems and levonorgestrel-containing contraceptives with oral or subdermal administration route.
      ]. Across the tested dosages, AUC0-24,ss, Cmax,ss and Cmin,ss values increased with increasing dose. As expected, in all dose groups Cmax,ss, Cav and Cmin,ss were considerably higher than those observed after 21 days of oral administration of 0.030 mg LNG (884, 327 and 184 pg/mL, respectively) [
      • Hofmann BM
      • Apter D
      • Bitzer J
      • Reinecke I
      • Serrani M
      • Höchel J
      • et al.
      Comparative pharmacokinetic analysis of levonorgestrel-releasing intrauterine systems and levonorgestrel-containing contraceptives with oral or subdermal administration route.
      ]. A previous study evaluated serum LNG concentrations during 7-year use of an LNG subdermal implant, and found that no pregnancies occurred in subjects with LNG levels >180 pg/mL [
      • Sivin I
      • Wan L
      • Ranta S
      • Alvarez F
      • Brache V
      • Mishell DR
      • et al.
      Levonorgestrel concentrations during 7 years of continuous use of Jadelle contraceptive implants.
      ]. This value has been regarded as an efficacy threshold level for LNG [
      • Sivin I
      • Wan L
      • Ranta S
      • Alvarez F
      • Brache V
      • Mishell DR
      • et al.
      Levonorgestrel concentrations during 7 years of continuous use of Jadelle contraceptive implants.
      • Westhoff CL
      • Chen BA
      • Jensen JT
      • Barnhart K
      • Thomas M
      • Teal S
      • et al.
      Phase I/II pharmacokinetic and pharmacodynamic evaluation of two levonorgestrel-only contraceptive patches in a multicenter randomized trial.
      ]. In our study, mean LNG concentrations on the PK-sampling day were above this threshold level, as well as most individual values. Only some subjects in the 0.095 and 0.115 mg groups had Cmin,ss values below 180 pg/mL but none of the subjects in the 0.135 mg group. A limitation of our study, designed to correlate ovulation incidence with LNG dosage, is that the low number of ovulations and the performance of PK sampling only in a subset of participants do not allow for valid correlation of ovulation incidence with LNG exposure, attempting to define a threshold exposure level for ovulation inhibition. Another limitation of our study is that we did not measure trough LNG concentrations at each visit. This would have provided information on the course of LNG levels during the treatment period and would have been an objective measure of compliance.
      A decrease of SHBG concentrations by approximately 60% was also observed in a previous PK study on oral administration of 0.150 mg LNG for 21 days, while 20% decrease was seen after 21-day oral administration of 0.030 mg LNG [
      • Kuhnz W
      • Al-Yacoub G
      • Fuhrmeister A.
      Pharmacokinetics of levonorgestrel in 12 women who received a single oral dose of 0.15 mg levonorgestrel and, after a washout phase, the same dose during one treatment cycle.
      ,
      • Hofmann BM
      • Apter D
      • Bitzer J
      • Reinecke I
      • Serrani M
      • Höchel J
      • et al.
      Comparative pharmacokinetic analysis of levonorgestrel-releasing intrauterine systems and levonorgestrel-containing contraceptives with oral or subdermal administration route.
      ].
      Lower SHBG levels, higher LNG clearance and lower LNG exposure in women with high BMI were also observed in other PK studies with oral and non-oral LNG-containing preparations [
      • Westhoff CL
      • Chen BA
      • Jensen JT
      • Barnhart K
      • Thomas M
      • Teal S
      • et al.
      Phase I/II pharmacokinetic and pharmacodynamic evaluation of two levonorgestrel-only contraceptive patches in a multicenter randomized trial.
      ,
      • Hofmann BM
      • Apter D
      • Bitzer J
      • Reinecke I
      • Serrani M
      • Höchel J
      • et al.
      Comparative pharmacokinetic analysis of levonorgestrel-releasing intrauterine systems and levonorgestrel-containing contraceptives with oral or subdermal administration route.
      • Praditpan P
      • Hamouie A
      • Basaraba CN
      • Nandakumar R
      • Cremers S
      • Davis A
      • et al.
      Pharmacokinetics of levonorgestrel and ulipristal acetate emergency contraception in women with normal and obese body mass index.
      ]. It is known that the efficacy of LNG treatment may be reduced in obese women due to lower exposure [
      • Westhoff CL
      • Chen BA
      • Jensen JT
      • Barnhart K
      • Thomas M
      • Teal S
      • et al.
      Phase I/II pharmacokinetic and pharmacodynamic evaluation of two levonorgestrel-only contraceptive patches in a multicenter randomized trial.
      ,
      • Praditpan P
      • Hamouie A
      • Basaraba CN
      • Nandakumar R
      • Cremers S
      • Davis A
      • et al.
      Pharmacokinetics of levonorgestrel and ulipristal acetate emergency contraception in women with normal and obese body mass index.
      • Kapp N
      • Abitbol JL
      • Mathé H
      • Scherrer B
      • Guillard H
      • Gainer E
      • et al.
      Effect of body weight and BMI on the efficacy of levonorgestrel emergency contraception.
      ]. In our study, the lower LNG exposure in subjects with BMI ≥30 kg/m2 did not seem to result in differences in ovarian activity, but the number of obese women was small. Studies with larger numbers of obese women will need to confirm that the high dosage of this new LNG-POP is sufficient for complete ovulation inhibition and adequate contraceptive efficacy in both obese and non-obese women.
      The median numbers of bleeding or spotting days during LNG treatment were lower than during 56-day treatment with desogestrel 0.075 mg [
      • Duijkers IJM
      • Heger-Mahn D
      • Drouin D
      • Skouby S.
      A randomised study comparing the effect on ovarian activity of a progestogen-only pill (POP) containing desogestrel and a new POP containing drospirenone in a 24/4 regimen.
      ]. However, the duration of the current study was too short to draw valid conclusions on the bleeding profile. With the desogestrel POP, the number of bleeding/spotting days decreased during longer treatment [
      • Palacios S
      • Colli E
      • Regidor P-A.
      A multicenter, double-blind, randomized trial on the bleeding profile of a drospirenone-only pill 4 mg over nine cycles in comparison with desogestrel 0.075 mg.
      ]. A study of longer duration needs to be performed to investigate if the higher-dose LNG-POP leads to an improved bleeding pattern compared to currently available POPs.
      In conclusion, increasing the dosage of the LNG-POP to 0.115 mg or 0.135 mg LNG per day resulted in consistent inhibition of ovulation. The higher dosages were safe and well-tolerated, and mean E2 concentrations during treatment were sufficient for prevention of hypoestrogenic side effects. The higher dose of 0.135 mg per day led to more pronounced suppression of follicular growth and serum estradiol concentrations, with smaller interindividual variation, as compared to 0.115 mg per day and will therefore reduce the risk of ovulation in case of forgotten tablets or intake delays, which are very common in real life [
      • Klipping C
      • Duijkers I
      • Trummer D
      • Marr J.
      Suppression of ovarian activity with a drospirenone-containing oral contraceptive in a 24/4 regimen.
      • Hou MY
      • Hurwitz S
      • Kavanagh E
      • Fortin J
      • Goldberg AB.
      Using daily text-message reminders to improve adherence with oral contraceptives: a randomized controlled trial.
      ]. Furthermore, the higher dosage might partly compensate for the anticipated lower exposure in obese women, who constitute a large proportion of the population. Future studies will need to investigate the contraceptive efficacy and bleeding profile of this new LNG-POP.

      Acknowledgments

      Financial support for the conduct of the study and preparation of the article was provided by Navad Life Sciences. The sponsor was involved in the study design, in the analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.

      Appendix B. Supplementary materials

      Appendix A. Laboratory methods

      Analyses of serum levonorgestrel (LNG) concentrations were performed by ACC GmbH Analytical Clinical Concepts, Leidersbach, Germany. The analytical method was validated in accordance with the requirements of the Committee for Medicinal Products for Human Use Guideline on Bioanalytical Method Validation and the current Food and Drug Administration (FDA) Guidance for Industry on Bioanalytical Method Validation, and Good Laboratory Practice principles [

      Committee for Medicinal Products for Human Use. Guideline on bioanalytical method validation. EMEA/CHMP/EWP/192217/2009 Rev.1 Corr. 2 (2011).

      US Department of Health and Human Services, Food and Drug Administration. Bioanalytical method validation. Guidance for industry (2018).

      ]. The method is based on liquid chromatography coupled to mass spectrometry (LC-MS/MS) with electrospray ionisation in the positive mode and multiple reaction monitoring. LNG-d6 was used as internal standard (ISTD). The lower limit of quantification of the method was 30.0 pg/mL, the upper limit of quantification was 5000.0 pg/mL, a linear relationship between concentration and signal intensity (given as peak area ratio analyte/ISTD) was obtained. Calibration of analyte was done by establishing linear regression functions after 1/x weighting of analyte/ISTD peak area ratio versus analyte concentration relationships. During pre-study validation all guideline-based criteria regarding within-run and between-run precision and accuracy had been fulfilled as well as validation of sample dilution. Also, recovery was in line with the acceptance criteria. Selectivity investigations including interference check, carryover, matrix effect (including influence of haemolysed or hyperlipemic plasma) were successfully realized. Initial validation was done on a Sciex Triple Quad 5500 detector connected with a CTC Analytics PAL hlTC-xt autosampler. The method was transferred to a second LC-MS/MS system (10L), also consisting of a Sciex Triple Quad 5500 detector connected with a CTC Analytics PAL HTC-xt autosampler with additional investigation of linearity and limits of quantitation as well as within-run precision and accuracy. Validation included freeze-thaw stability, storage at room temperature as well as long-term stability over the maximum duration of sample storage.
      Analyses of estradiol (E2), progesterone (P), sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were performed by Synlab Pharma Analytics & Services Germany GmbH, using chemiluminescent microparticle immunoassays (Abbott) on the automated analyzer Architect Plus i2000SR (Abbott).
      E2 with a calibration range of 34 to 1000 pg/mL and P with a calibration range of 1.43 – 40 ng/mL, as pharmacodynamic endpoints for determination of ovulation inhibition, and SHBG with a calibration range from 7.5 to 250 nmol/L underwent a comprehensive pre-study validation in accordance with the current FDA Guidance for Industry on Bioanalytical Method Validation [

      US Department of Health and Human Services, Food and Drug Administration. Bioanalytical method validation. Guidance for industry (2018).

      ]. The pre-study validations fulfilled all requirements and furthermore, adequate stability over the sample storage period was demonstrated. For FSH and LH, validation followed the requirements of the manufacturer of the immunoassay as well as internal standard operating procedures.

      References

        • McCann MF
        • Potter LS.
        Progestin-only oral contraception: a comprehensive review.
        Contraception. 1994; 50: S9-195
        • World Health Organization
        Medical eligibility criteria for contraceptive use: a WHO family planning cornerstone.
        5th ed. World Health Organization, Geneva2015
        • Moghissi KS.
        Morphologic changes in the ovaries of women treated with continuous microdose progestogens.
        Fertil Steril. 1972; 23: 739-744
        • Moghissi KS
        • Syner FN
        • McBride L.
        Contraceptive mechanism of microdose norethindrone.
        Obstet Gynecol. 1973; 41: 585-594
        • Landgren B-M
        • Balogh A
        • Shin MW
        • Lindberg M
        • Diczfalusy E.
        Hormonal effects of the 300 µg` norethisterone (NET) minipill. 2. Daily gonadotropin levels in 43 subjects during a pretreatment cycle and during the second month of NET administration.
        Contraception. 1979; 20: 585-605
        • Landgren B-M
        • Diczfalusy E.
        Hormonal effects of the 300 µg norethisterone (NET) minipill. 1. Daily steroid levels in 43 subjects during a pretreatment cycle and during the second month of NET administration.
        Contraception. 1980; 21: 87-113
        • Spona J
        • Schneider WHF
        • Ch Bieglmayer
        • Schroeder R
        • Pirker R.
        Ovulation inhibition with different doses of levonorgestrel and other progestogens: clinical and experimental investigations.
        Acta Obstet Gynecol Scand. 1979; : 7-15
        • Rice C
        • Killick S
        • Hickling D
        • Coelingh Bennink H
        Ovarian activity and vaginal bleeding patterns with a desogestrel-only preparation at three different doses.
        Hum Reprod. 1996; 11: 737-740
        • Klipping C
        • Duijkers I
        • Remmers A
        • Faustmann T
        • Zurth C
        • Klein S
        • et al.
        Ovulation-inhibiting effects of dienogest in a randomized, dose-controlled pharmacodynamic trial of healthy women.
        J Clin Pharmacol. 2012; 52: 1704-1713
        • Rice CF
        • Killick SR
        • Dieben T
        • Coelingh Bennink H
        A comparison of the inhibition of ovulation achieved by desogestrel 75 µg and levonorgestrel 30 µg daily.
        Hum Reprod. 1999; 14: 982-985
        • Duijkers IJM
        • Heger-Mahn D
        • Drouin D
        • Skouby S.
        A randomised study comparing the effect on ovarian activity of a progestogen-only pill (POP) containing desogestrel and a new POP containing drospirenone in a 24/4 regimen.
        Eur J Contracept Reprod Health Care. 2015; 20: 419-427
        • Collaborative Study Group on the Desogestrel-containing Progestogen-only Pill
        A double-blind study comparing the contraceptive efficacy, acceptability and safety of two progestogen-only pills containing desogestrel 75 µg/day or levonorgestrel 30 µg/day.
        Eur J Contracept Reprod Health Care. 1998; 3: 169-178
        • Mishell DR
        • Lumkin M.
        Initial clinical studies of intravaginal rings containing norethindrone and norgestrel.
        Contraception. 1975; 12: 253-260
        • Faundes A
        • Brache de Mejias V
        • Leon P
        • Robertson D
        • Alvarez F.
        First year clinical experience with six levonorgestrel rods as subdermal contraception.
        Contraception. 1979; 20: 167-175
        • Oettel M
        • Kochhar PS
        • Osterwald H
        • Zarapkar MS
        • Walawalkar R.
        The Progestin-Only Pill (POP) is not a niche option: new preclinical and clinical data about the interrelations between levonorgestrel-dose, peripheral as well as central contraceptive effects and bleeding behaviour.
        J Reproduktionsmed Endokrinol. 2015; 12: 1-5
        • Hoogland HJ
        • Skouby SO.
        Ultrasound evaluation of ovarian activity under oral contraceptives.
        Contraception. 1993; 47: 583-590
        • Sivin I
        • Wan L
        • Ranta S
        • Alvarez F
        • Brache V
        • Mishell DR
        • et al.
        Levonorgestrel concentrations during 7 years of continuous use of Jadelle contraceptive implants.
        Contraception. 2001; 64: 43-49
        • Westhoff CL
        • Chen BA
        • Jensen JT
        • Barnhart K
        • Thomas M
        • Teal S
        • et al.
        Phase I/II pharmacokinetic and pharmacodynamic evaluation of two levonorgestrel-only contraceptive patches in a multicenter randomized trial.
        Contraception. 2018; 98: 151-157
        • Schindler AE
        • Campagnoli C
        • Druckmann R
        • Huber J
        • Pasqualini JR
        • Schweppe KW
        • et al.
        Classification and pharmacology of progestins.
        Maturitas. 2003; 46: S7-S16
        • Taubert H-D
        • Kuhl H.
        Kontrazeption mit Hormonen, ein Leitfaden für die Praxis.
        2nd ed. Georg Thieme Verlag, Stuttgart, New York1995: 84
        • Brache V
        • Blumenthal PD
        • Alvarez F
        • Dunson TR
        • Cochon L
        • Faundes A.
        Timing of onset of contraceptive effectiveness in Norplant® implant users.
        Contraception. 1999; 59: 245-251
        • Barbieri RL.
        Hormone treatment of endometriosis: the estrogen threshold hypothesis.
        Am J Obstet Gynecol. 1992; 166: 740-745
        • Riggs MM
        • Bennetts M
        • van der Graaf PH
        • Martin SW.
        Integrated pharmacometrics and systems pharmacology model-based analysis to guide GnRH receptor modulator development for management of endometriosis.
        CPT Pharmacometrics Syst Pharmacol. 2012; 1: e11
        • Hadji P
        • Colli E
        • Regidor P-A.
        Bone health in estrogen-free contraception.
        Osteoporos Int. 2019; (Published online)https://doi.org/10.1007/s00198-019-05103-6
        • Kuhnz W
        • Al-Yacoub G
        • Fuhrmeister A.
        Pharmacokinetics of levonorgestrel in 12 women who received a single oral dose of 0.15 mg levonorgestrel and, after a washout phase, the same dose during one treatment cycle.
        Contraception. 1992; 46: 443-454
        • Hofmann BM
        • Apter D
        • Bitzer J
        • Reinecke I
        • Serrani M
        • Höchel J
        • et al.
        Comparative pharmacokinetic analysis of levonorgestrel-releasing intrauterine systems and levonorgestrel-containing contraceptives with oral or subdermal administration route.
        Eur J Contracept Reprod Health Care. 2020; 25: 417-426
        • Praditpan P
        • Hamouie A
        • Basaraba CN
        • Nandakumar R
        • Cremers S
        • Davis A
        • et al.
        Pharmacokinetics of levonorgestrel and ulipristal acetate emergency contraception in women with normal and obese body mass index.
        Contraception. 2017; 95: 464-469
        • Kapp N
        • Abitbol JL
        • Mathé H
        • Scherrer B
        • Guillard H
        • Gainer E
        • et al.
        Effect of body weight and BMI on the efficacy of levonorgestrel emergency contraception.
        Contraception. 2015; 91: 97-104
        • Palacios S
        • Colli E
        • Regidor P-A.
        A multicenter, double-blind, randomized trial on the bleeding profile of a drospirenone-only pill 4 mg over nine cycles in comparison with desogestrel 0.075 mg.
        Arch Gynecol Obstet. 2019; 300: 1805-1812
        • Klipping C
        • Duijkers I
        • Trummer D
        • Marr J.
        Suppression of ovarian activity with a drospirenone-containing oral contraceptive in a 24/4 regimen.
        Contraception. 2008; 78: 16-25
        • Hou MY
        • Hurwitz S
        • Kavanagh E
        • Fortin J
        • Goldberg AB.
        Using daily text-message reminders to improve adherence with oral contraceptives: a randomized controlled trial.
        Obstet Gynecol. 2010; 116: 633-640
      1. Committee for Medicinal Products for Human Use. Guideline on bioanalytical method validation. EMEA/CHMP/EWP/192217/2009 Rev.1 Corr. 2 (2011).

      2. US Department of Health and Human Services, Food and Drug Administration. Bioanalytical method validation. Guidance for industry (2018).