Title: Effects of depot medroxyprogesterone acetate, the copper IUD and the levonorgestrel
implant on testosterone, sex hormone binding globulin and free testosterone levels: Ancillary
study of the ECHO* randomized clinical trial
Version: 6 Date: 29 Jun 2023
Reviewer's report:
This paper describes the evaluation and the findings of the ancillary study  from ECHO study
which are quite important for public health.
The original ECHO multi-centre RCT,  evaluated primarily the effect of  type of contraceptive
method (DMPA injection, Copper IUD and Levornorgestrel implant) on differences in HIV
acquisition among women 16-35 years old who were  previously uninfected with HIV.
The current ancillary paper used the serum samples stored in East London, South Africa site to
evaluate the effect of type of contraceptive method on the changes in the levels of Testosterone,
SHBG  and Free Testosterone values (between baseline and at 6 months ). The authors evaluated
the outcomes using  paired the values for the same individuals  between the two time points.
GENERAL COMMENTS
By nature of it being an ancillary study based on one site, and being limited to those participant
with available outcomes at both baseline and at 6 months, this sub-study no longer benefits the
advantages of  an RCT that the parent ECHO study had.
1. There would be a need to compare the distributions of sociology-demographic/economic and
other relevant baseline factors between the three groups of contraceptive methods. Authors have
tried to show this in Table 1 in terms age, education, income, parity and menses, but it would
be interesting to  see the role of other factors as well  to the extent of the data availability on
these. Examples include but not limited to weight/BMI, medications and medical history, as the
authors find relevant.  Table 1 could in addition include   p-values to show how statistically
comparable or different at baseline the groups are with respect to these factors.
2. I would expect to see the univariate and multivariable regression model(s) for the estimates
of T, SHBG and  free T values before and after accounting for potential confounders. The
description of the model and the final multivariable model model building strategy  should be
added in the Methods section.
3. Furthermore,  authors have mentioned use of  mixed effects model  in the methods section,
which is great, but it is unclear which factors would be in apriori be considered as random and
which would form  fixed effects in this model.
SPECIFIC COMMENTS
1. FLOWCHART  (PAGE 11):  Would the 135 women  assessed for hormone levels at 6 months
under the IUD arm include any of the 7  who did not receive the IUD ?  I propose that none of
the of the 7 with no IUD should form part of the 135 assessed in this arm. Please clarify and
kinndly ensure the numbers in the 3 arms are adding up well.
2. BASELINE TABLE (page 12): please  refer to the general comment in (1)
3. TABLE 3 (Page 13): On Hormonal results (IR)
(a) Please label the columns titles adequately.
For example:
-  Col 3 (labeled "Baseline") could be clearer if named "Baseline Median  (IR)"
-  Col 5:  (labeled "6 months") could be clearer if named "6 month Median   (IR)"
-  Col 7:  (labeled "Change") could be clearer if named "Median change (6mo vs
Baseline)  (IR)" .  Do please note that median change is not same as change in
Median.
4. For lines 232-255: While results have been presented in terms of crude median (IR), I have
not seen the results of the mixed level regression that the authors indicated they would use in the
Methods section. I suggest that  the results of these models are presented in a tabular format and
taking into considerations the suggestions proposed above. The authors should consider using
these models as long as the relevant assumptions are tested and are approximately met. The
methods the authors use to test the assumptions for the  relevant models should be added in the
methods sections.