outlines the derivation of the analysis sample for measurement of prolactin, breast milk volume and clinical parameters. This includes 222 HIV-positive women, 97 randomly assigned to group A and 125 assigned to group B, as well as 24 HIV-negative women (all in group B).
Flowchart of HIV-infected women who contributed blood prolactin and breast milk volume measurements by group randomization assignment and actual mode of feeding practice.
These were derived from the full cohort of 297 HIV-positive women (146 and 151 randomized to group A and B, respectively) with live-births occurring between the beginning of the study (May 2001) and March 2003. Among the 146 HIV-positive women assigned to group A, 97 (66.4%) had data available for this analysis, 14 (9.6%) did not attend the 4 or 4.5-month visit and 35 (24.0%) attended the visit but the samples were not collected either because the participant reported not having yet stopped breastfeeding (n = 22) or for other reasons. Among the 151 HIV-positive women assigned to group B, 125 (82.8%) had data available for this analysis, 16 (10.6%) did not complete the relevant postnatal study visit and 10 (6.6%) attended the visit but the samples were not collected. There were no systematic differences between the women for whom data were available in group A versus group B with regard to demographic, clinical or disease stage parameters.
Among the 97 HIV-positive women assigned to the early weaning group, 92 (95%) reported having stopped all breastfeeding before the post-weaning measurements were taken (one woman assigned to the continued breastfeeding group also stopped breastfeeding). Postweaning samples were obtained for 83 (85.6%) women at the scheduled 4.5-month visit, for 11 (11.3%) at the 5-month visit, and for three at a later time point. All except one of the 125 women in group B were still breastfeeding when the samples were obtained: 113 (90.4%) at the 4.5-month visit, 10 (8.0%) at the 5-month visit and two at a later visit. For clarity, the analysis was based on actual reported practice rather than group assignment. Results were essentially unchanged if an intent-to-treat analysis was performed (data not shown).
There were no significant differences in maternal age, parity, plasma viral load, CD4 cell count or body mass index (BMI) between the HIV-positive women who had weaned compared with those who were still breastfeeding at the time of measurement. Women who weaned were significantly more likely to have breast engorgement and to report fever since the last visit in comparison with women who continued to breastfeed ().
Characteristics of the 222 HIV-positive and 24 HIV-negative women included in the analysis by actual breastfeeding practices at the time of measurement of prolactin and breast milk volume.
Prolactin and pumped breast milk volume as a measure of weaning compliance
Baseline plasma prolactin levels (just prior to pumping) were significantly lower in women who claimed to have weaned in comparison with women who reported continued breastfeeding providing confirmation that weaning had occurred. Prolactin levels measured after breast milk pumping increased to a similar extent in both groups (), but the percentage of women with very high prolactin levels was far greater among the breastfeeding women, presumably in response to the suckling stimulus of breast milk pumping (data not shown). There were no differences between HIV-positive and HIV-negative women who were still breast feeding.
The volume of breast milk pumped in 10 min was significantly less among women who had weaned in comparison with those still breastfeeding (). Among the 93 women who had weaned, 17.2% pumped no detectable milk, and 22.6% pumped less than 5 ml. By comparison only 4.7% of 129 women who were still breastfeeding pumped no milk and 9.3% pumped < 5ml (P < 0.008).
Characteristics of 71 HIV-positive women with post-pump viral loads.
Clinical signs and duration of weaning
There were no significant differences in prolactin or breast milk volume by duration of time since cessation of breastfeeding. Although, there was a trend towards higher baseline prolactin with a short interval (< 7 days), and lower breast milk volume with a longer interval (> 14 days) since breastfeeding cessation, these differences did not reach statistical significance. There were also no significant differences by the duration of the weaning period ().
Factors associated with basal prolactin level (pre-pumping) and breast milk volume among 93 HIV-positive women who had stopped all breastfeeding by the time of measurement.
We found a non-significant increase in the volume of breast milk that was pumped among women with breast engorgement or breast pain and a significant increase in women who reported feeling ill or fever since weaning. Prolactin was significantly elevated among those who reported fever since breastfeeding ended (). There were no associations between prolactin and breast milk volume and either maternal age, parity, CD4 cell count or body mass index.
Exclusive breastfeeding and prolactin levels
All the women who had not stopped breastfeeding reported both day and night-time breast feeds over the previous 2 weeks and eight of 129 (6.2%) reported giving the child something other than breast milk over the same period. The median baseline prolactin levels were higher in those 121 women who reported only exclusive breastfeeding [median, 112; interquartile range (IQR), 79-161] compared with the eight women who reported mixed breastfeeding in the previous 2 weeks (median, 68; IQR, 47-110), but this did not reach statistical significance (P = 0.09). Post-pumping prolactin levels and pumped volumes were similar among exclusive and non-exclusive breastfeeding women.
Resumption of menses
Resumption of menses by 6 months post-partum was significantly more common among women who stopped breastfeeding than those who continued to breastfeed (). Among those who had stopped all breastfeeding, basal prolactin levels post-weaning were significantly lower (mean 36.9 ng/ml) among those who subsequently resumed menses by 6 months than those who did not resume menses by this time (mean 75.3 ng/ml) (P = 0.01). Post-pumping prolactin levels were also lower but the difference did not reach significance. Basal and post-pumping prolactin levels among those who were still breastfeeding were also significantly lower among those who subsequently resumed menses.
Breast milk viral load in a random subset of 71 women
To conserve resources, we randomly selected breast milk samples from 80 women (40 who had stopped breastfeeding and 40 who were still breastfeeding) among the 222 evaluable subjects to measure breast milk viral load. Thirty-one of the 40 who had weaned contained a sufficient quantity of breast milk (≥1 ml) for viral load testing; whereas all 40 women who continued to breastfeed had sufficient quantity for testing (). Detectable HIV RNA was measured in 21 of 31 (67.7%) women who had stopped breastfeeding compared with 17 of 40 (42.5%) women who were still breastfeeding (P = 0.03). The median RNA concentrations (if detectable) were 7930 copies/ml in those who had stopped and 904 copies/ml in those who were still breastfeeding (P = 0.005) (). HIV RNA concentrations > 1000 copies/ml were found among 45.2% of those who had stopped versus 12.5% of those who were still breastfeeding (P = 0.002).
Breast milk HIV viral load among pumped breast milk from 31 women who recently weaned compared with breast milk from 40 lactating women of the same postnatal age.
We were also able to compare breast milk viral load longitudinally (i.e. before and after weaning) among 29 of 31 women who had weaned. The pre-weaning sample was obtained at the visit when breastfeeding was due to stop, on average 2 weeks prior to the post-weaning measurement. Although a similar proportion of women, 18 of 29 (62.1%), had detectable virus before weaning as after weaning, the median pre-weaning breast milk viral load (if detectable) was only 353 copies/ml, and only 17.2% had > 1000 copies/ml. Breast milk viral load rose significantly from the pre-weaning sample to the postweaning sample (P = 0.001); a median increase of 15 822 copies/ml (38 to > 750 000 copies/ml). No increase in breast milk viral load occurred over an equivalent 2-week interval among women still breastfeeding at 5 months.
Among those who had stopped breastfeeding, there was a highly significant correlation between breast milk viral load and (1) baseline plasma prolactin (r 0.578; P = 0.0007); (2) post-pumping prolactin (r = 0.363; P = 0.04); and (3) a nearly significant correlation = with breast milk volume pumped (r 0.330; P 0.06). None of these three parameters was= associated with = breast milk viral load from the same women obtained prior to weaning 2 weeks earlier.
In the group with viral load measurements, 18 women reported that the duration of weaning was less than 2 days and 12 women reported that its duration was 2-7 days. None reported the weaning process lasting beyond 7 days. This information was missing for one person. There was no significant difference in post-weaning viral load among women who stopped within 2 days (median 313 copies/ml) versus those who stopped less rapidly (2-7 days; median 646 copies/ml). Although mastitis and reported fever were more common among women who had stopped breastfeeding (), we found no association between breast milk viral load and these two parameters but numbers were small.