The injectable contraceptives depot medroxyprogesterone acetate and norethisterone enanthate substantially and differentially decrease testosterone and sex hormone binding globulin levels: A secondary study from the WHICH randomized clinical trial

Chanel Avenant; Mandisa Singata-Madliki; Alexis J Bick; Donita Africander; Yusentha Balakrishna; Karl-Heinz Storbeck; Johnson M Moliki; Sigcinile Dlamini; Salndave Skosana; Jenni Smit; Mags Beksinska; Ivana Beesham; Ishen Seocharan; Joanne Batting; George J Hofmeyr; Janet P Hapgood

doi:10.1371/journal.pone.0307736

. 2024 Aug 23;19(8):e0307736. doi: 10.1371/journal.pone.0307736

The injectable contraceptives depot medroxyprogesterone acetate and norethisterone enanthate substantially and differentially decrease testosterone and sex hormone binding globulin levels: A secondary study from the WHICH randomized clinical trial

Chanel Avenant ^1,^#, Mandisa Singata-Madliki ^2,^#, Alexis J Bick ¹, Donita Africander ³, Yusentha Balakrishna ⁴, Karl-Heinz Storbeck ³, Johnson M Moliki ¹, Sigcinile Dlamini ¹, Salndave Skosana ¹, Jenni Smit ⁵, Mags Beksinska ⁵, Ivana Beesham ⁵, Ishen Seocharan ⁴, Joanne Batting ², George J Hofmeyr ^2,^6,^7,^‡, Janet P Hapgood ^1,^8,^‡,^*

Editor: Renee Ridzon⁹

¹Department of Molecular and Cell Biology, University of Cape Town, Cape Town, South Africa

²Effective Care Research Unit, Eastern Cape Department of Health/Universities of the Witwatersrand and Fort Hare, East London, South Africa

³Department of Biochemistry, Stellenbosch University, Stellenbosch, South Africa

⁴Biostatistics Research Unit, South African Medical Research Council, Durban, South Africa

⁵Wits MRU (MatCH Research Unit), Department of Obstetrics and Gynecology, Faculty of Health Sciences, University of the Witwatersrand, Durban, South Africa

⁶Walter Sisulu University, East London, South Africa

⁷Department of Obstetrics and Gynecology, University of Botswana, Gaborone, Botswana

⁸Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa

⁹National Institute of Allergy and Infectious Diseases, UNITED STATES OF AMERICA

Competing Interests: The authors have declared that no competing interests exist.

‡ GJH and JPH also contributed equally to this work.

^✉

* E-mail: Janet.Hapgood@uct.ac.za

Contributed equally.

Roles

Chanel Avenant: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing

Mandisa Singata-Madliki: Conceptualization, Funding acquisition, Resources, Writing – review & editing

Alexis J Bick: Investigation, Writing – review & editing

Donita Africander: Writing – original draft, Writing – review & editing

Yusentha Balakrishna: Data curation, Formal analysis, Writing – review & editing

Karl-Heinz Storbeck: Methodology, Writing – review & editing

Johnson M Moliki: Investigation, Writing – review & editing

Sigcinile Dlamini: Investigation, Writing – review & editing

Salndave Skosana: Methodology

Jenni Smit: Resources, Writing – review & editing

Mags Beksinska: Resources, Writing – review & editing

Ivana Beesham: Resources, Writing – review & editing

Ishen Seocharan: Data curation

Joanne Batting: Resources, Writing – review & editing

George J Hofmeyr: Conceptualization, Writing – original draft, Writing – review & editing

Janet P Hapgood: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing

Renee Ridzon: Editor

PMCID: PMC11343371 PMID: 39178280

Abstract

HIV acquisition risk with norethisterone (NET) enanthate (NET-EN) is reportedly less than for depo-medroxyprogesterone acetate intramuscular (DMPA-IM). We investigated the effects of these progestin-only injectable contraceptives on serum testosterone and sex hormone binding globulin (SHBG) levels, since these may play a role in sexual behavior and HIV acquisition. The open-label WHICH clinical trial, conducted at two sites in South Africa from 2018–2019, randomized HIV-negative women aged 18–40 years to 150 mg DMPA-IM 12-weekly (n = 262) or 200 mg NET-EN 8-weekly (n = 259). We measured testosterone by UHPLC-MS/MS and SHBG by immunoassay in matched pairs of serum samples collected at baseline (D0) and at peak serum progestin levels at 25 weeks post initiation (25W) (n = 214–218 pairs). Both contraceptives substantially decreased, from D0 to 25W, the total testosterone [DMPA-IM D0 0.560, 25W 0.423 nmol/L, -24.3% (p < 0.0001); NET-EN D0 0.551, 25W 0.253 nmol/L, -54.1%, (p < 0.0001)], SHBG [DMPA-IM D0 45.0, 25W 32.7 nmol/L, -29.8% (p < 0.0001); NET-EN D0 50.2, 25W 17.6 nmol/L, -65.1% (p < 0.0001)], and calculated free testosterone levels [DMPA-IM D0 6.87, 25W 5.38 pmol/L, -17.2% (p = 0.0371); NET-EN D0 6.00, 25W 3.70, -40.0% (p < 0.0001)]. After adjusting for change from D0, the total testosterone, SHBG and calculated free testosterone levels were significantly higher for DMPA-IM than NET-EN (64.9%, p < 0.0001; 101.2%, p < 0.0001; and 38.0%, p = 0.0120, respectively). The substantial and differential decrease in testosterone and SHBG levels does not explain our previous finding of no detected decrease in risky sexual behavior or sexual function for DMPA-IM or NET-EN users from D0 to 25W. Medroxyprogesterone (MPA) and NET are androgenic and are both present in molar excess over testosterone and SHBG concentrations at 25W. Any within or between contraceptive group androgenic effects on behavior in the brain are likely dominated by the androgenic activities of MPA and NET and not by the decreased endogenous testosterone levels. The clinical trial was registered with the Pan African Clinical Trials Registry (PACTR 202009758229976).

Introduction

Progestin-only injectable contraceptives, mainly depo-medroxyprogesterone acetate intramuscular (DMPA-IM), are the most common contraceptive methods in sub-Saharan Africa [1–3], which has a high incidence and prevalence of HIV, particularly among young women and girls [4]. Meta-analyses of higher quality observational clinical data reported a significant 40–50% increased risk of HIV acquisition with DMPA-IM compared to no hormonal contraception, unlike for limited data on NET-EN [5, 6]. Head-to-head comparisons of HIV risk among women using DMPA-IM versus NET-EN indicated a potential 32–41% increase in HIV risk for DMPA-IM users versus NET-EN users [5, 7, 8]. The Evidence for Contraceptive Options and HIV Outcomes (ECHO) randomized trial comparing DMPA-IM, the copper intra uterine device (IUD) and Jadelle, a levonorgestrel (LNG)-containing implant, did not detect a significant difference in HIV acquisition risk of 50% or more between these methods [9]. However, the ECHO trial data do not inform on the risk for HIV infection for DMPA-IM compared to NET-EN, or for DMPA-IM compared to no contraception or irregular use of condoms [9]. Obtaining robust data on the effects of DMPA-IM and NET-EN on factors that potentially affect risk for HIV acquisition is important for understanding their risks and benefits and the biological mechanisms thereof. The Women’s Health, Injectable Contraception and HIV (WHICH) trial, an open-label clinical trial that randomized women to DMPA-IM or NET-EN, investigated differences in hormonal, psychological, behavioral, menstrual and immune effects within and between the two contraceptives. While both contraceptives substantially and similarly reduced estradiol to postmenopausal levels one week after the 6-month injection, the data suggested more sexual exposure to HIV with DMPA-IM than NET-EN [10].

Levels of endogenous sex steroid hormones are likely to play a role in multiple physiological pathways and health outcomes, including susceptibility to sexually transmitted infections and HIV, immune function and sexual behavior [11]. While estradiol is known to be protective against HIV acquisition in the female genital tract [11], the role of androgens [12, 13] in HIV acquisition is unknown. Circulating levels of sex hormones reportedly modify cellular morphology in the brain [14] and influence higher brain functions such as cognition, memory and mood [15]. Indeed, decreased testosterone levels are associated with several undesired effects such as increased headaches, mood changes, and reduced sexual desire and libido [16]. Thus, decreased testosterone levels may decrease libido resulting in less exposure to HIV. Evidence on the effects of libido is, however, contentious as some women on combined oral contraception (COC) had decreased testosterone levels without a decrease in libido [17]. It is also unclear whether sexual function in women is associated with endogenous androgen concentrations due to insufficient robust data and uncertainty relating to the sensitivity and specificity of androgen quantification assays in some studies [18]. In addition, hormonal contraception is also associated with several androgenic effects including acne, hirsutism, weight gain, androgenic alopecia, unfavourable lipid profiles, and diabetes [19–23]. DMPA-IM has been linked to increased incidence of type 2 diabetes, oily skin and acne in women [20, 22–24], while very little information is available on the androgenic effects of NET-EN in women. Furthermore, it is unknown to what extent any androgenic effects may be attributed to the known androgenic activity of MPA and NET via the androgen receptor (AR) in vitro [25].

Testosterone is one of the major androgens in the serum of premenopausal women [26, 27]. However, clinical studies investigating the effects of progestin-only contraceptives on testosterone levels are limited, and the available studies mainly assessed effects in women using COCs which result in reduced total serum testosterone concentrations (reviewed in [12]). Earlier studies showed decreased serum testosterone levels in postmenopausal breast cancer patients after administration of oral MPA [28]. Similarly, it has been shown that subcutaneous DMPA (DMPA-SC) [29] and the LNG implant [30] both decrease testosterone levels in premenopausal women. A decrease in testosterone was also detected in premenopausal women (3–4 women) and in a transgender population [31] administered DMPA-IM, while a significant decrease in testosterone was not detected in postpartum women administered NET-EN [32]. To understand the clinical significance of changes in testosterone levels in women, it is important to have accurate data on the effects of progestins not only on total testosterone levels, but also that of free testosterone and sex hormone binding globulin (SHBG) levels. Total testosterone includes both the biologically inactive, circulating, SHBG-bound testosterone, as well as the biologically active testosterone circulating either free (not bound to plasma protein) or that weakly bound to albumin [33]. Studies to date suggest that the effects of progestins on testosterone and SHBG are dependent on the type of contraceptive. For instance, ethinyl estradiol combined with either LNG or drospirenone (DRSP) reduced total and free testosterone levels, but increased SHBG levels [34]. This increase in SHBG levels is likely due to ethinyl estradiol as it is known to increase hepatic SHBG production (reviewed in [35]). In contrast, the progestin-only injectable DMPA-SC is associated with a significant decrease in total testosterone and SHBG, but not in free testosterone levels [29]. To our knowledge, only two other non-comparative studies have shown that the injectable contraceptives DMPA-IM [36] and NET-EN [37] decrease SHBG levels.

There is a lack of robust data on the effects of DMPA-IM and NET-EN on testosterone and SHBG levels, as well as their relative effects. Furthermore, no data are available for the effects of these contraceptives at peak serum progestin levels. In this study, we compared the serum levels of testosterone and SHBG, as well as changes in the levels of total and calculated free testosterone and SHBG, within and between two arms of the WHICH trial randomizing women to DMPA-IM or NET-EN, at peak progestin levels.

Methods

Primary study, ethics and biosafety

This study is a secondary study from the open-label randomized WHICH clinical trial. The primary aims of the trial were estradiol levels and menstrual, psychological and behavioral measures relevant to HIV risk. The WHICH study protocol and primary study have been reported elsewhere [10]. The study was registered retrospectively with the Pan African Clinical Trials Registry (PACTR 202009758229976 https://pactr.samrc.ac.za/Search.aspx). All women provided informed, written consent to authorize study participation and storage of samples. The study adhered to the ethical principles outlined in the Declaration of Helsinki (World Medical Association, 2011) and the Constitution of the Republic of South Africa (Bill of Rights). Ethical approval for the secondary study conducted at the University of Cape Town (UCT) was obtained from the UCT Faculty of Health Sciences Human Research Ethics Committee (HREC REF no. 664/2018). The authors did not have access to information that could identify individual participants during or after data collection.

Study design and sample collection

Briefly, HIV-negative young women (18–40 years) seeking contraception at the East London and Mdantsane public health clinics and hospitals (Frere and Cecilia Makiwane Hospitals), South Africa (331 participants), and the research site of the MatCH Research Unit (MRU), University of the Witwatersrand, based in Durban, KwaZulu-Natal, South Africa (189 participants) were randomized to 150 mg DMPA-IM 12-weekly or 200 mg NET-EN 8-weekly. Exclusion criteria were participants who received DMPA-IM in the previous 6 months or NET-EN in the previous 4 months, were living with HIV, or were using or intending to use medication which might have interfered with biological measurements such as steroids or drugs affecting renal function such as pre-exposure prophylaxis drugs (for HIV). Participants were recruited and followed from 5 November 2018 to 30 November 2019. We screened 546 and randomized 521 women to DMPA-IM (262) and NET-EN (259). A total of 86.9% (n = 453) completed a 25-week study visit with a similar number completing in both method groups.

Blood samples were collected at baseline (D0) and at 25 weeks (25W), i.e. about one week after the 6-month NET-EN (the 4^th NET-EN injection) or DMPA-IM (the 3^rd DMPA-IM) injection, and serum was separated and stored at -80°C.

Total testosterone measurements

These measurements were performed between 2 January 2020 and 31 December 2022. Testosterone was quantified by ultra-high performance liquid tandem mass spectrometry (UHPLC-MS/MS) on stored serum samples from WHICH study participants at D0 and 25W, as described in S1 Appendix. Testosterone data were obtained for 214 and 215 participants, at D0 and 25W respectively, from the DMPA-IM arm, with D0 data for one participant being absent for technical reasons. Testosterone data were obtained for 219 and 218 participants at D0 and 25W, respectively from the NET-EN arm. Accuracy (% BIAS) and precision (% CV) were both less than 15% at all concentrations tested (0.05, 0.1, 0.5, 5 and 50 ng/mL) (S1 Table in S1 File). Recovery (% Extraction efficiency) and matrix effects were both within acceptable limits (S1 Table in S1 File). The limit of detection (LOD) and lower limit of quantification (LLOQ) for testosterone were 0.0250 ng/mL (0.0870 nmol/L) and 0.0500 ng/mL (0.173 nmol/L), respectively, while the upper limit of quantification (ULOQ) was 50.0 ng/mL (173.37 nmol/L) (S2 Table in S1 File). A linear calibration curve was obtained between the LLOQ and ULOQ (R² > 0.996). All researchers performing the assays were blinded.

SHBG measurements and calculation of free testosterone

These measurements were performed between 2 January 2020 and 31 December 2020. SHBG was measured on stored serum samples from WHICH study participants at D0 and 25W by means of chemiluminescent microparticle immunoassay (CMIA) (Abbott Laboratories) (sensitivity level of quantification (LOQ) 0.02 nmol/L; no detectable cross-reactivity). All researchers performing the assays were blinded. Free testosterone was calculated according to the method of Vermeulen et al. [38]. SHBG data were obtained for 217 and 216 participants, at D0 and 25W respectively, from the DMPA-IM arm, with 25W data for one participant being absent for technical reasons. SHBG data were obtained for 219 and 218 participants at D0 and 25W, respectively, from the NET-EN arm, with 25W data for one participant being absent for technical reasons. Free testosterone was calculated for all those participants for whom SHBG and testosterone data was available (214 and 215 for D0 and 25W, respectively in DMPA-IM arm; 219 and 217 for D0 and 25W, respectively, for NET-EN arm).

Data analysis

UHPLC-MS/MS data collection and analysis were performed using MassLynx 4.2 (Waters Corporation). The ratio of the analyte peak area to internal standard peak area was determined for all the calibration curve samples, internal quality controls (IQCs) and serum samples. Testosterone values below the LLOQ, but above the LOD were assigned 0.5 x LLOQ (n = 16 D0; n = 30 25W), while values lower than LOD were assigned as 0.000 (n = 5 25W). For SHBG all the values were above the level of quantification (LOQ) (0.02 nmol/L).

We performed a modified intention-to-treat (mITT) analysis on the whole cohort and a per protocol (PP) analysis on a subgroup of the whole cohort. To obtain a subgroup for the PP analysis, we used UHPLC-MS/MS data obtained for study (MPA and NET) and non-study (LNG, nestorone, etonogestrel and gestodene) progestin levels at D0 and 25W in donor-matched serum samples [39]. For the testosterone and SHBG subgroup PP analysis, we excluded results from all women (104/436 participants, or 23.8%) that had any non-study serum progestin at concentrations ≥ 1.5 nM, at either D0 or 25W, from the mITT group. In addition to the above-mentioned non-study progestins, for the PP analysis we also excluded women in the DMPA-IM arm that had NET concentrations greater than 1.5 nM, at either D0 or 25W, as well as women in the NET-EN arm that had MPA concentrations higher than 1.5 nM, at either D0 or 25W. In total 23.8% (104/346 participants) of the women were excluded in the PP analysis.

Results in Tables 2–4 and S3 Table in S1 File were analysed using Stata version 16 (College Station, TX: StataCorp LLC). For total testosterone and SHBG, D0 and 25W values were nmol/L, while pmol/L values for free testosterone were used. Shapiro-Wilks normality tests indicated that all hormonal data were not normally distributed, and hence data are expressed as median with interquartile range (IQR) (Table 2 and S3 Table in S1 File). A mixed-effects linear regression model was fitted for each of the (natural) log-transformed outcomes (total testosterone, SHBG, free testosterone). Random effects to account for repeated measures within participants and to account for clustering by site were included in the model. A model coefficient, β, on the log-scale can be back-transformed using e^β. To facilitate the interpretation of the results, we report the percentage changes on the original scale, calculated as (e^β – 1) × 100%. Thus, the mean differences between D0 and 25W as well as between allocated arms are presented as percentages (back-transformed coefficients) with 95% confidence intervals (CIs) (Tables 3 and 4). Fig 2 and S1 Fig in S1 File were generated using GraphPad Prism 9.31 from GraphPad Software, Inc. (La Jolla California, USA), while statistical differences determined by mixed-effects linear regression model, as mentioned above, are shown. All results were considered significant for p < 0.05.

Table 2. Total testosterone (nmol/L), SHBG (nmol/L) and free testosterone (pmol/L) outcomes at baseline and 25 weeks (mITT analysis).

	DMPA-IM		NET-EN
	Median (IQR)	n	Median (IQR)	n
Total Testosterone (nmol/L)
D0	0.560 (0.354; 0.815)	214	0.551 (0.350; 0.853)	219
25W	0.423 (0.281; 0.610)	215	0.253 (0.086; 0.385)	218
Change (25W - D0)	-0.119 (-0.288; 0.003)		-0.291 (-0.513; -0.090)
SHBG (nmol/L)
D0	45.0 (33.6; 68.8)	217	50.2 (35.1; 72.4)	219
25W	32.7 (24.8; 44.4)	216	17.6 (12.2; 22.9)	218
Change (25W - D0)	-12.2 (-25.1; -2.3)		-32.3 (-51.5; -17.7)
Free Testosterone (pmol/L)
D0	6.87 (2.81; 13.82)	214	6.00 (2.00; 14.0)	219
25W	5.38 (2.28; 11.73)	215	3.70 (0.87; 9.33)	217
Change (25W - D0)	-0.71 (-5.48; 1.33)		-1.50 (-7.02; 1.16)

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IQR (25th and 75th Percentile)

Table 4. Pairwise comparisons of hormonal results for DMPA-IM vs NET-EN expressed as mean percentage differences (95% CI) (mITT analysis).

	Mean percentage difference between DMPA-IM and NET-EN at D0		Mean percentage difference between DMPA-IM and NET-EN at 25W		Mean percentage difference at 25W between DMPA-IM and NET-EN, after adjusting for baseline
	DMPA_D0 –NET-EN_D0		DMPA_25W –NET-EN_25W
	Mean % (95% CI)*	p-value^*	Mean % (95% CI)^*	p-value^*	Mean % (95% CI)^*	p-value^*
Total Testosterone	-2.9 (-13.6; 9.3)	0.6347	60.3 (42.4; 80.4)	<0.0001	64.9 (46.1; 86.1)	<0.0001
SHBG	-5.6 (-14.6; 4.3)	0.2556	89.9 (71.8; 109.9)	<0.0001	101.2 (82.2; 122.3)	<0.0001
Free testosterone	-0.2 (-21.9; 27.5)	0.9867	37.7 (7.6; 76.2)	0.0110	38.0 (7.3; 77.4)	0.0120

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*estimates and p-values from mixed effects linear regression, accounting for repeated measurements per participant and clustering by site.

Table 3. Mean percentage changes (95% CI) in total testosterone, SHBG and free testosterone (mITT analysis).

	DMPA-IM	NET-EN	DMPA-IM vs NET-EN
	Mean (95% CI)^*	Mean (95% CI)^*	p-value^*
Total Testosterone
% Change from D0	-24.3 (-30.5; -17.5)	-54.1 (-57.9; -50.0)	<0.0001
SHBG
% Change from D0	-29.8 (-34.6; -24.7)	-65.1 (-67.5; -62.6)	<0.0001
Free Testosterone
% Change from D0	-17.2 (-30.7; -1.1)	-40.0 (-49.8; -28.4)	0.0120

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* estimates and p-values from mixed effects linear regression, accounting for repeated measurements per participant and clustering by site. Note, mixed effects linear regression indicated that both DMPA-IM and NET-EN significantly decreased total testosterone (p < 0.0001 for both), SHBG (p < 0.0001 for both) and free testosterone (DMPA-IM p = 0.0371; NET-EN p < 0.0001) levels from D0 to 25W.

Fig 2 — Graphs indicate median with interquartile range (IQR). Significant differences were calculated by mixed effects linear regression, accounting for repeated measurements per participant and clustering by site and are indicated by asterisks where * and **** represent p<0.05 and p<0.0001, respectively.

Results

Primary data and baseline characteristics

Of 521 participants enrolled, results are reported for all matched serum samples available from the whole cohort for 435 (83%) participants both at baseline and at peak (mITT analysis). The trial profile is shown in Fig 1.The excluded participants in the mITT analysis include 11 (2%) that became HIV positive, 6 (1%) that became pregnant, 65 (12%) that were lost to follow up (i.e., those that did not provide a 25W blood sample) and 5 participants that had a missing blood result for either SHBG or for testosterone, due to technical problems with the sample.

Baseline data is shown in Table 1.

Table 1. Baseline characteristics of women by randomization method^$ (mITT analysis).

	Baseline (D0)
	DMPA-IM		NET-EN
		n		n
Age, years: Mean (SD)	25 (4.8)	217	24.9 (4.7)	219
Ethnicity		217		219
Xhosa	145 (66.8)		148 (67.6)
Zulu	67 (30.9)		71 (32.4)
Mixed race	1 (0.5)		0 (0.0)
Other African ethnicity	4 (1.8)		0 (0.0)
Previous use of method ^#		217		219
DMPA-IM	161 (74.2)		160 (73.1)
NET-EN	69 (31.8)		65 (29.7)
Marital status		217		219
Single	211 (97.2)		213 (97.3)
Married	6 (2.8)		6 (2.7)
Highest level of education		217		219
Primary school, complete	2 (0.9)		4 (1.8)
High school, not complete	88 (40.6)		76 (34.7)
High school, complete	82 (37.8)		95 (43.4)
Post high school education	45 (20.7)		44 (20.1)
Source of income		217		219
Unemployed	183 (84.3)		192 (87.7)
Employed	34 (15.7)		27 (12.3)

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^$ Unless indicated otherwise, values represent n-value (%)

^#Prior to exclusion period, numbers given are for those that responded, and in brackets are % of those that responded. Note that some participants reported using both contraceptive methods prior to the exclusion period.

DMPA-IM and NET-EN decrease total and free testosterone and SHBG concentrations

At baseline, the median total testosterone levels in the DMPA-IM and NET-EN arms were 0.560 nmol/L and 0.551 nmol/L, respectively (Table 2 and Fig 2). Both DMPA-IM and NET-EN significantly decreased total testosterone levels from D0 to 25W by 24.3% (p < 0.0001) and 54.1% (p < 0.0001), respectively (Table 3), with median concentrations at 25W in the DMPA-IM and NET-EN arms being 0.423 nmol/L and 0.253 nmol/L, respectively (Table 2). A significant difference in total testosterone levels between arms was detected at 25W with DMPA users having a 60.3% higher testosterone level than NET-EN users (p < 0.0001) (Table 4). No significant difference in total testosterone levels between arms was detected at D0 (Table 4). A significant difference was detected in total testosterone levels at 25W between arms, after adjusting for baseline, with DMPA-IM users showing a 64.9% (p < 0.0001) higher total testosterone level than NET-EN users (Table 4).

At baseline the median SHBG levels in the DMPA-IM and NET-EN arms were 45.0 nmol/L and 50.2 nmol/L, respectively (Table 2 and Fig 2). Both DMPA-IM and NET-EN significantly reduced SHBG levels from D0 to 25W by 29.8% (p < 0.0001) and 65.1% (p < 0.0001), respectively (Table 3), with median SHBG levels at 25W in the DMPA-IM and NET-EN arms being 32.7 nmol/L and 17.6 nmol/L, respectively (Table 2 and Fig 2). A significant difference in SHBG levels between arms was detected at 25W with DMPA users having an 89.9% higher SHBG level than NET-EN users (p < 0.0001) (Table 4). At D0 no significant difference in SHBG levels was detected between the two arms (Table 4). After adjusting for the change from D0, DMPA-IM users had 101.2% (p < 0.0001) higher SHBG levels than NET-EN users at 25W (Table 4).

Median calculated free testosterone concentrations at baseline in the DMPA-IM and NET-EN arm were 6.87 pmol/L and 6.00 pmol/L, respectively (Fig 2 and Table 2). At 25W the median free testosterone levels in the DMPA-IM and NET-EN arms were 5.38 pmol/L and 3.70 pmol/L, respectively (Table 2). Both DMPA-IM and NET-EN significantly reduced free testosterone levels from D0 to 25W by 17.2% (p = 0.0371) and 40.0% (p < 0.0001), respectively (Table 3). Additionally, a significant difference in free testosterone levels between arms was detected at 25W with DMPA users having a 37.7% higher free testosterone level than NET-EN users (p = 0.0110) (Table 4). At D0 no significant difference in free testosterone levels was detected between the two arms (Table 4). After adjusting for the change from D0, DMPA-IM users had 38.0% (p = 0.0120) higher free testosterone levels at 25W than NET-EN users (Table 4).

In a subgroup PP analysis, after excluding for non-study progestins, significant differences were detected for the same comparisons for the whole cohort (mITT analysis) compared to the subgroup (PP analysis), for total testosterone, SHBG and free testosterone concentrations (S3 Table and S1 Fig in S1 File). The baseline characteristics for the subgroup of women randomized to DMPA-IM or NET-EN used in the PP analysis are shown in S4 Table in S1 File.

Discussion

We report for the first time on the effects of DMPA-IM and NET-EN on total and free testosterone and SHBG levels at peak progestin levels from a randomized trial. We detected substantial decreases from D0 to 25W in measured total testosterone (-24.3% and -54.1%) and SHBG levels (-29.8% and -65.1%) and calculated free testosterone levels (-17.2% and -40.0%), for DMPA-IM and NET-EN users, respectively. Whether the lower median concentrations of total testosterone at 25W could be classified as hypoandrogenic or post-menopausal, is unclear from the literature [27, 40, 41]. When comparing the mean percentage difference in change between these contraceptives from D0 to 25W, we report that DMPA-IM use results in a significantly smaller change in total testosterone, SHBG levels and free testosterone than NET-EN use.

To our knowledge there is no published literature to allow direct comparison between our results for DMPA-IM and NET-EN at peak progestin concentrations. One observational study reported values for DMPA-IM that are 27-fold greater and 2.5-fold lower [42] than the values we determined for total and free testosterone, respectively. A study among 15 women using the 104 mg DMPA-SC injection reported a significant decrease in total testosterone and SHBG, measured by immunoassay, but not for calculated free testosterone, one week after the 3-month DMPA-SC injection [29]. Their values are about 2–4-fold higher than our median total testosterone values, but comparable to our median SHBG values and about 5–6-fold lower than our values calculated for free testosterone. Very little information is available for NET-EN, but one study reported a reduction in SHBG levels five days after NET-EN injection, with levels of SHBG 2.3-fold higher than our value at 25W [37]. Possible reasons for differences between our results and other results for DMPA-IM or NET-EN include lower power due to small sample sizes, confounding factors due to non-randomization, and differences in sampling times and methods of testosterone quantification. Our use of UHPLC-MS/MS is likely to have generated more accurate and lower values for testosterone than immunoassay methods [43, 44]. Nevertheless, our results, reviewed together with limited published data [45], suggest that most progestin-only contraceptives significantly and substantially decrease SHBG levels, and that DMPA-IM decreases total and calculated free testosterone levels to a greater extent than DMPA-SC at peak serum MPA levels, while both have and similar effects on SHBG levels.

The biological, behavioral and clinical consequences of substantially decreased levels of total and free serum testosterone and SHBG for both DMPA-IM and NET-EN are difficult to interpret. When comparing effects on sexual behavior between DMPA-IM and NET-EN, we have reported more sexual activity and more risky sexual behavior and possibly more exposure to HIV with DMPA-IM than NET-EN [10]. This is consistent with a greater decrease in total testosterone levels for NET-EN relative to DMPA-IM, as testosterone may be associated with increased libido. However, while the relative effects of these contraceptives are important, the individual effects from baseline to 25W are also biologically and clinically relevant. If increased testosterone results in increased sexual activity in women, one would expect a decrease in sexual behavior within both arms. However, a substantial decrease in sexual behavior from baseline to 25W was not detected in the WHICH cohort for either contraceptive [10].

An important potential confounding factor in understanding the effects of DMPA-IM and NET-EN on androgenic effects is that both these progestins are themselves androgenic. They have similar binding affinities for and potencies via the AR in vitro when compared head-to-head to each other and dihydrotestosterone [25, 46], although one study reported that MPA is less potent than testosterone and dihydrotestosterone [47]. While some studies have reported that NET has more androgenic activity than MPA in animal pre-clinical models [48], there is no robust data on androgenic activity of DMPA-IM and NET-EN in women. It is not possible to extrapolate these in vitro and animal data directly to relative androgenic activities in women due to multiple confounding factors, including metabolism, cross-talk with other pathways and species-, gene- and cell-specific effects. We have reported that the medium peak serum concentrations for MPA and NET in DMPA-IM and NET-EN users are 6.6 and 14 nmol/L, respectively, in these WHICH trial samples [39]. Notably, these concentrations are much higher than those for endogenous total or calculated free testosterone [DMPA-IM 0.423 nmol/L and 5.38 pmol/L; NET-EN 0.253 nmol/L and 3.70 pmol/L, respectively] as well as those of estradiol (25W DMPA-IM 77 pmol/L; 25W NET-EN 70 pmol/L) [10]. Thus, it is possible that any androgenic effects of DMPA-IM and NET-EN are dominated by the androgenic properties of MPA and NET themselves, rather than the relatively very low but differential levels of testosterone reported here, and postmenopausal but similar levels of estradiol for both contraceptives [10].

One potential confounding factor in our randomized study is misreporting of DMPA-IM or NET-EN use, or use of contraceptives containing estrogens such as COCs, before and/or during the trial. We and others have reported that it is common for women not to self-report non-study progestin use before initiation and during clinical trials on contraception [39, 49]. We performed a PP analysis of the testosterone and SHBG data on a subgroup of participants after excluding those with concentrations of non-study progestins above 1.5 nM at either D0 or 25W. These PP analyses resulted in the same significant differences compared to the mITT results, and comparable median values for total and free testosterone and SHBG, suggesting that non-study progestin use did not introduce significant bias to these values. Our testosterone and SHBG findings are unlikely to be confounded by differences in baseline characteristics between contraceptive arms, given the stringent randomization process.

Conclusions

Total and free testosterone and SHBG potentially affect multiple physiological pathways and clinical outcomes. Our published findings of increased risky sexual behavior for DMPA-IM relative to NET-EN users are consistent with the differential decrease in total endogenous testosterone levels. However, our lack of detection of a general decrease in risky sexual behavior from D0 to 25W for either contraceptive is not consistent with a substantial decrease in endogenous testosterone levels being the major determinant of changes in sexual behavior. Taken together, it is likely that progestins themselves are the major determinants of androgenic effects, including on sexual behavior in the brain, that potentially affect HIV acquisition risk for DMPA-IM and NET-EN, as well as their relative effects. Understanding these complex mechanisms requires more research.

Supporting information

S1 File. S1 Fig and S1 to S4 Tables.

(DOCX)

pone.0307736.s001.docx^{(662.6KB, docx)}

S1 Appendix. Appendix 1 testosterone quantification.

(DOCX)

pone.0307736.s002.docx^{(23.6KB, docx)}

Acknowledgments

We thank all the women who participated in the WHICH clinical trial. We thank Karen van der Merwe for project administration and assistance in preparation and submission of the manuscript. We thank Marietjie Stander and Erick van Schalkwyk for assistance with the UHPLC-MS/MS assays.

Data Availability

"The raw data is available without restriction and has been deposited in the BioStudies public database. The data is publicly available at https://www.ebi.ac.uk/biostudies/studies/S-BSST1403 or DOI 10.6019/S-BSST1403."

Funding Statement

This work was supported by the U.S. National Institutes of Health and South African Medical Research Council through its U.S.-SA Program for Collaborative Biomedical Research (R01HD083026 [NICHD & NIAID] and R01AI152118 [NIAID]) to J.P.H. (https://www.nih.gov & https://www.samrc.ac.za), and a UCT Vice Chancellor’s Advancing Womxn award to J.P.H (https://uct.ac.za). The clinical trial was funded by the South African Medical Research Council Grants, Innovation and Product Development (SAMRC N/A grant number) grant to M.S-M (https://www.samrc.ac.za/innovation/grants-innovation-and-product-development). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0307736.r001

Decision Letter 0

Renee Ridzon

9 Feb 2024

PONE-D-23-20153The injectable contraceptives depot medroxyprogesterone acetate and norethisterone enanthate substantially and differentially decrease testosterone and sex hormone binding globulin levels: a secondary study from the WHICH randomized clinical trial.PLOS ONE

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Thank you for your submission and please see reviews below. It has been exceedingly difficult to find reviewers so apologize for delay in return of this and in fact there were invitations to over 25 people to act as a reviewer for this manuscript. This is a substudy of measurements of hormones from the larger WHICH study. There is reference to findings of the larger study multiple times and the results of this study are helpful to put context around the data presented here. Unfortunately, the references are designated as "submitted" so the reader is not able to see that study. This led me to wonder if it would be better to publish this one after the publication of the main study. Given the time that has passed, this main study may be slated for publication which would be ideal.

Lines 54-55 Will the reader understand what the abbreviations D0 and 25W mean? Would it be clearer to say in the beginning of the sentence that there was a substantial decrease from baseline to week 25 (if I am understanding what these abbreviations mean) and then give the % decline in the values. It appears that the decrease is calculated as 25W-D0 and the decrease is expressed as a negative %. A negative decrease (seems like a double negative) could be interpreted as an increase. If not, would be clearer to say that the decrease is a % (without the negative sign) or that the value at 25W is x% lower than D0? This comment pertains to the results section as well. If this is the standard way this information is expressed, please disregard the comment.

Line 59-The % difference (decrease) for testosterone and SHBG is expressed without a leading negative sign. Why is the difference expressed as a positive number here and negative above? Is there a reason why both a decrease in median level and a mean percentage difference is given?

Line 64-The time period of measurement expressed here appears to be D0 to 24W whereas above it is D0 and 25W. Is this correct? Why are these times different and why the is the formatting different for the presentation of the number of weeks (24W vs W25)?

Line 119- Is the implication here that decreased levels of testosterone impact susceptibility to HIV though a decreased libido that leads to less exposure or is it another mechanism? Should this be explained further here?

Line 152-It might be interesting to tell the reader whether it is the bound or the free testosterone that is active (or the different activities of these 2 moieties), if there is room, especially as it is later stated how the different hormones impact, free, bound and total testosterone.

Line 188-Presumably this is an open label study due the difference in the dosing interval of the agents. It would be helpful to state this.

Line 196-Are the PrEP drugs referred to here for HIV? That should be stated.

Line 258-It is stated that a subgroup was used for the PP analysis and that this group excluded of persons who used “non-study progestin >1.5”. It is very unclear what this is and is it possible that examples be given?

Line 298-Why are the numbers 219 and 217 listed in the white lines of this table under “previous use of method” whereas in the other parts of this table these totals are in the gray lines? “Previous use of method” should be reworded to “Previous method used”. Since the numbers for this category add to greater than the total for each arm, need to tell the reader that some participants used both of the methods listed. Why are Single and Married listed under this heading?

Line 306-Usually, the p value is listed after the 2 values being compared.

Line 342-Perhaps state for clarity “DMPA-IM and NET-EN significantly reduced median SHBG levels from baseline to 25W by -27% and -66%, respectively (Table 3)". Is there a reason that the levels are presented in an inconsistent manner here? For example, the SHBG levels are presented as values in nmol/L with a difference expressed in concentration in nmol/L but the SHBG is presented as a negative % with the difference presented as % change.

Line 347-“Median free testosterone concentrations at baseline and 25W were not significantly different between the two arms at baseline (p = 0.842)” Does this mean that all 4 of the measurements were not different from each other? Or does that mean there was not a difference between baseline and 25W in each of the 2 arms? Please clarify the statement. The statement seems to contradict the next sentence of “At 25W the 349 median free testosterone level in the DMPA-IM arm of 5.00 pmol/L was significantly 350 higher than 4.00 pmol/L in the NET-EN arm (p = 0.0124)”. This entire paragraph is hard to followed and needs to be rewritten for clarity.

Line-358-Does data need to be presented for both medians and means? If so why? Since it is stated that results were similar for medians and means, it is not clear if this information is needed.

Line 374-The discussion seems a bit long and could be shortened with the discussion on other studies made more terse.

Line 376-It is stated “significant decreases from D0 to 25W in measured total testosterone (-24% and -54%)”. The decrease is a percent, not a negative percent. By saying that there is a decrease from baseline to 25W, it is understood that the value is lower, the % expresses the magnitude of the decrease. Adding the negative sign seems to make this confusing.

Line 431-Does 4/5 and 3/5 mentioned here refer to markers of sexual behavior? If so, perhaps reword to “no significant differences were detected for 4/5 markers in 432 the DMPA-IM arm and for 3/5 markers in the NET-EN arm”.

Line 437-Sometimes HIV is used and sometimes HIV-1 is used, as here. Is there a reason for this difference?

Line 438-Data on sexual desire is not included in this paper and the discussion should be focused on the data from this study. That there was a difference in sexual desire is noted in the WHICH cohort is mentioned on Line 429 and that seems sufficient and the discussion here should focus on this paper’s data, especially since other paper is not yet published.

Line 444-The statistical power of the WHICH study is not the subject of this discussion and no information has been given to the reader to support claims about the power of the study on sexual behavior results.

Line 460-Can it be stated that data have been reported when the paper has not yet been published?

Line 481-What is the basis for saying that the study is high-powered? No information on the power of the study is given in the methods. How could there be misreporting of DMPA-IM or NET-EN use when these are the interventions that are being examined in this study and presumably being administered in a controlled manner?

Line 490-stated “we would have had to exclude a large proportion of the women (for use of non-study progestins) and substantially reduced the power of this study” What are these and how many of the participants used them? Was it possible to exclude such women from the study? If not, why not?

Line 506-“Our findings of increased risky sexual behavior for DMPA-IM relative to NET-EN users” no data on this has been presented and this should not be part of the conclusions of this study. Further is stated that it is likely that progestins are determinants of androgenic effects. Given that it is also stated that a large number of the women in this study may have used non-study progestins, does this introduce significant bias?

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: No

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General comments:

This manuscript reports a secondary analysis from a clinical trial that explored the differences in serum testosterone and sex hormone binding globulin levels between two injectable contraceptives.

My main concern regards the statistical methods. The methods seem to lack coherence. For instance, I found it confusing why the authors claim non-parametric tests are required for all data, but then use methods that assume normality. There may also be methods used that are not reported in the methods section.

Also, I will point out that the choice of running a statistical model is more about whether the residuals are normally distributed and not the outcome itself. Linear models are somewhat robust against deviations from normality. Looking at figure 2, I think analyzing a log-transformed outcomes is feasible. I would much prefer if you were able to fit a single statistical model for each of outcome and then evaluate differences between groups and time points. That would reduce the risk of type I errors and allow for much more coherent methods and results sections.

Although I am here to evaluate design and methods, I strongly suggest reducing the introduction and probably the discussion. In the vast majority of papers, the introduction can be 3-4 paragraphs, i.e., (1) broad background on problem, (2) specifics on problem to be addressed, and (3) objectives and brief synopsis of design and methods.

Thus, I don't feel the paper presently meets criterion 3 (Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail) and criterion 5 (The article is presented in an intelligible fashion and is written in standard English) since it's wordy. Though, I think you could fix these.

Specific comments:

1. (lines 51-52) If you are using paired tests, this should be the sample size of the pairs.

2. (lines 54-60) Confidence intervals or p-values are needed for all of these comparisons.

3. (lines 225-226) This must be described somewhere in the manuscript especially since the information has not been published. There is no word count for PLOS ONE, so you should feel free to include this information here.

4. In lines 267-269, you note that all data are not normally distributed but then in lines 274-277 you use a model that assumes normality. Granted, you log-transformed your outcomes for the mixed models. Why can't that be done for the simple tests as well?

5. (Table 1, line 295, line 306, …) Claiming "similarity" is incorrect when using superiority tests. These statements suggest that the absence of evidence indicates evidence of absence. You can claim no difference, but not similarity. Statements like this need to be revised since a p>0.05 doesn't necessarily mean there were no differences. Significance testing for baseline imbalance in randomized trials been has regarded as unnecessary (see Altman DG. Comparability of randomised groups. The Statistician 1985; 125-136; Senn, S. Testing for baseline balance in clinical trials. Statistics in Medicine 1994; 1715-1726). I recommend removing the significance testing entirely from table 1.

6. (Table 3, line 315) I don't recall something in the methods describing analyses of percentage change.

7. (Table 4) I encourage you to report your effects on the scale in which they are analyzed.

8. In addition, you use the word "similar" in other situations, e.g., lines 363 and 366. Similar is vague and subjective and I suggest being specific about the comparisons made in these statements.

Reviewer #2: thanks for the opportunity to review this well written paper

A few minor comments only

Line 103 – please clarify what you mean by the “6 month injection” as neither of these are 6 month injections.

Please explain line 105 further

Line 196 – assume you mean PrEP for HIV - clarify

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Reviewer #2: Yes: Katherine Gill

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PLoS One. 2024 Aug 23;19(8):e0307736. doi: 10.1371/journal.pone.0307736.r002

Author response to Decision Letter 0

23 Apr 2024

Thanks to the editor and the reviewers for all the very constructive suggestions and queries. We have attended to all the comments.

A detailed point-by-point response can be found in the uploaded file "response to reviewers".

Attachment

Submitted filename: Responses to Reviewers.docx

pone.0307736.s003.docx^{(37KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0307736.r003

Decision Letter 1

Renee Ridzon

16 May 2024

PONE-D-23-20153R1The injectable contraceptives depot medroxyprogesterone acetate and norethisterone enanthate substantially and differentially decrease testosterone and sex hormone binding globulin levels: a secondary study from the WHICH randomized clinical trial.PLOS ONE

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Additional Editor Comments:

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Reviewers' comments:

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

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6. Review Comments to the Author

Reviewer #1: Thank you for considering my comments to the initial draft. To follow up on a couple of these comments:

Significance testing in table 1:

The main point of my comment is to provide a more accurate assessment of baseline imbalance. It is not about transparency. For example, two groups with small sample sizes will likely have p>0.05 for all characteristics. So, then having a smaller sample size is better…? No, it's because p-values are a poor assessment of baseline comparability since the p-value is driven by the sample size. Other methods, such as standardized mean differences, provide a better assessment of differences. I still believe this should be changed.

Normality assumption:

I'm fine with reporting medians and IQRs for summary statistics, but in Table 2 you are reporting medians and IQRs right next to p-values from regression models that assume normality. This remains confusing for me and I think has the potential for misinterpretation. If someone takes a quick glance at this, I fear s/he will either (1) think the summary statistics are means and 95% CIs or, more likely, think the p-value are for a difference in medians. I encourage you to report what is actually being tested next to the p-value to avoid this potential for confusion.

Additional comments:

- Please provide details on the back-transformation mentioned in line 258 since this is reported in the text and a table.

- (Table 3) For free testosterone, why is the DMPA-IM vs. NET-EN p-value < 0.05 when the two 95% CIs overlap?

(lines 299-301 and Table 4) It's not clear to me what the values in this table mean. For total testosterone, is the -2.9 a difference at baseline? Or is it a mean percentage change? It's not clear from the title or the notes what these values are.

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PLoS One. 2024 Aug 23;19(8):e0307736. doi: 10.1371/journal.pone.0307736.r004

Author response to Decision Letter 1

29 Jun 2024

Thank you for the comments.

The response is in the file "response to reviewers"

Attachment

Submitted filename: Responses to Reviewers.docx

pone.0307736.s004.docx^{(18KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0307736.r005

Decision Letter 2

Renee Ridzon

11 Jul 2024

The injectable contraceptives depot medroxyprogesterone acetate and norethisterone enanthate substantially and differentially decrease testosterone and sex hormone binding globulin levels: a secondary study from the WHICH randomized clinical trial.

PONE-D-23-20153R2

Dear Dr. Hapgood,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0307736.r006

Acceptance letter

Renee Ridzon

15 Jul 2024

PONE-D-23-20153R2

PLOS ONE

Dear Dr. Hapgood,

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. S1 Fig and S1 to S4 Tables.

(DOCX)

pone.0307736.s001.docx^{(662.6KB, docx)}

S1 Appendix. Appendix 1 testosterone quantification.

(DOCX)

pone.0307736.s002.docx^{(23.6KB, docx)}

Attachment

Submitted filename: Responses to Reviewers.docx

pone.0307736.s003.docx^{(37KB, docx)}

Attachment

Submitted filename: Responses to Reviewers.docx

pone.0307736.s004.docx^{(18KB, docx)}

Data Availability Statement

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PERMALINK

The injectable contraceptives depot medroxyprogesterone acetate and norethisterone enanthate substantially and differentially decrease testosterone and sex hormone binding globulin levels: A secondary study from the WHICH randomized clinical trial

Chanel Avenant

Mandisa Singata-Madliki

Alexis J Bick

Donita Africander

Yusentha Balakrishna

Karl-Heinz Storbeck

Johnson M Moliki

Sigcinile Dlamini

Salndave Skosana

Jenni Smit

Mags Beksinska

Ivana Beesham

Ishen Seocharan

Joanne Batting

George J Hofmeyr

Janet P Hapgood

Roles

Abstract

Introduction

Methods

Primary study, ethics and biosafety

Study design and sample collection

Total testosterone measurements

SHBG measurements and calculation of free testosterone

Data analysis

Table 2. Total testosterone (nmol/L), SHBG (nmol/L) and free testosterone (pmol/L) outcomes at baseline and 25 weeks (mITT analysis).

Table 4. Pairwise comparisons of hormonal results for DMPA-IM vs NET-EN expressed as mean percentage differences (95% CI) (mITT analysis).

Table 3. Mean percentage changes (95% CI) in total testosterone, SHBG and free testosterone (mITT analysis).

Fig 2. Total testosterone (nmol/L), SHBG (nmol/L) and free testosterone (nmol/L), outcomes at baseline and 25 weeks (mITT analysis).

Results

Primary data and baseline characteristics

Fig 1. Trial profile.

Table 1. Baseline characteristics of women by randomization method$ (mITT analysis).

DMPA-IM and NET-EN decrease total and free testosterone and SHBG concentrations

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Renee Ridzon

Roles

Author response to Decision Letter 0

Decision Letter 1

Renee Ridzon

Roles

Author response to Decision Letter 1

Decision Letter 2

Renee Ridzon

Roles

Acceptance letter

Renee Ridzon

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Table 1. Baseline characteristics of women by randomization method^$ (mITT analysis).