Folliculogenesis is a key aspect of female reproductive function. Disruption of this process leads to compromise in female fertility potential. In the course of folliculogenesis, several follicles are destroyed and end up as atretic follicles.10 This study made use of the rat model to study folliculogenesis, owing to the relatively short cycle length averaging 4.8 days.11
Findings from the histological analysis of the ovarian tissue of the rats used in this study revealed a reduction in the number of granulosa and basal cells of follicles across various stages of follicular development in the study groups when compared to the control group. This reduction was more marked in the group that received efavirenz. (This is as shown in Figs. 2–6). Published studies on the effect of HAART on reproductive functions have shown highly heterogeneous outcomes. While most of the studies have outlined the effect of antiretroviral medications on male reproductive functions, only a few have looked at the effect of antiretroviral medications on female reproductive functions. In the study conducted by Awodele and co-workers on rodents to demonstrate the effect of HAART on the reproductive functions of rodents, there was a reduction in follicular development as well as the levels of progesterone, prolactin, and estradiol in the female rodents that received HAART when compared to controls.12 This is consistent with findings from our study. However, the exact mechanism of this action has not been elucidated.
Awodele et al. found that there was a decrease in the weight of the testes and epididymis of the rodents given HAART when compared to controls. The study revealed that the sperm count and the motility of the rodents on HAART were also reduced when compared to the controls. Hormonal parameters in the rodents like FSH, LH, and testosterone were all reduced in the group that received HAART when compared to controls.12
Collazos et al studied the effect of HAART on reproductive hormones of patients living with HIV and showed that there was an increase in serum levels of estradiol and testosterone in patients receiving HAART, but levels of LH and FSH were not influenced.13 In our study, there was no significant change in the serum levels of LH, while the serum levels of FSH were elevated in the study group that had EFV in the combination. This elevation was statistically significant. While Collazos’ work was on human participants, this work was conducted in a rat model and this could account for the difference in findings. Additionally, Collazos did not take into account follicular development in his study.13
Moreno-Perez et al noted an association between protease inhibitors and the development of erectile dysfunction in HIV-infected males. This study did not state if this was linked to spermatogenesis.14. Collazos et al. demonstrated that the use of antiretroviral treatment was the only predictive variable for sexual dysfunction in a group of HIV-positive patients managed with antiretroviral therapy.13. The authors further stated however that sexual dysfunction was not related to hormonal factors in that study.13
In another paper, Savasi and co-workers demonstrated a higher proportion of sperm nuclear fragmentation in HIV-positive individuals on HAART when comparisons were made to HIV-infected individuals not receiving HAART.15 On the flip side in a subsequent study, Savasi et al. stated that the duration of use and type of antiretroviral medication did not significantly influence semen quality despite generally low sperm parameters in HIV-positive patients.16 The mean count of primary, antral, and secondary follicles was lower in the study groups than in the control group. FSH and LH are both gonadotropins and play crucial roles in follicular development.10 Awodele et al. (2018) demonstrated similar findings with a reduction in FSH and LH and disruption of follicular functions.2
In our study, serum levels of LH did not show any significant difference between the study groups and the control group. The analysis of the levels of FSH and LH between the study groups and the control revealed a significant degree of variation. FSH levels in the group that received TDF were reduced when compared to the control group. FSH levels were markedly elevated in the group that received the fixed-dose combination of the drugs when compared to the control group. This difference was statistically significant (p = 0.05). An elevated level of FSH is a significant marker of menopause as females in menopause demonstrate high levels of FSH due to marked reduction in follicular development and reduction in the level of estradiol. The implication of this is that there is no negative feedback of estrogen on FSH levels, hence the marked elevation in the level of FSH itself. The group that received the FDC containing EFV possibly demonstrated elevated levels of FSH due to the toxic effect of the medication on the ovaries, hence follicular damage giving rise to the unopposed action of estrogen and FSH elevation. Awodele and coworkers demonstrated similar findings.12
In our other treatment groups that received EFV, FSH was marginally elevated when compared to the control group. FSH was however elevated in the 3TC group when compared against controls, but this difference was not statistically significant (p = 0.08). FSH levels are known to correlate with ovarian failure and serum levels of estrogen. This relationship is inverse, as depletion of ovarian follicles leads to reduced levels of estrogen.
Histological findings of the group that received EFV, revealed a distortion of the granulosa and basal cells with marked reduction of all follicles as shown in Fig. 3. Figure 4 shows the histological findings of the group that received TDF, which demonstrates that there was a non-significant reduction in the granulosa and basal cells with follicles at various stages of maturation.
The group that received 3TC showed a marginal decrease in granulosa and basal cells with a less than normal distribution of follicles across various stages of maturation. The reduction in the number of follicles was more marked in the study group that received EFV when compared to the study groups that received TDF, 3TC, and a combination of all three drugs. The group that received a combination of EFV, TDF, and 3TC as a fixed-dose combination had granulosa and basal cells sparsely distributed. The follicles were markedly reduced across various stages of development. The effect was seen more in follicles at the antral stage of development. A decreased follicular count has been documented in female Wistar rats exposed to Heracleum persicum.17 This herbal medicinal plant is famously used in Iran for a variety of traditional medicinal purposes. This plant has been used in some traditional settings as a form of contraception. However, this is the first time that such changes have been observed in the context of HAART in this animal model.
AMH is a marker for reproductive potential and ovarian function and also a surrogate for follicular development. It is used for the estimation of ovarian reserve and a prediction of fertility potential. The correlation of AMH with the antral follicular count is because AMH is secreted by the granulosa cells of the developing follicle. As menopause is being approached, AMH values correlate with the number of antral follicles in the ovary. This has been demonstrated in mice as the antral follicular count is directly proportional to the levels of AMH.18 Changes in the level of AMH are known to occur relatively early as it relates to ovarian aging.
Furthermore, changes in AMH are known to predate changes in FSH levels and inhibin B.19 Thus, AMH seems a better predictor of ovarian reserve than Inhibin B and FSH. It also seems a more reliable marker of ovarian reserve, because it does not fluctuate through the menstrual cycle.19 Of note, the average AMH level in this study was lower in the study groups that received HAART with the combined antiretroviral therapy and EFV alone when compared to the control group, although the difference was not statistically significant. (p = 0.180). Recent studies tend to suggest that AMH could be protective against ovarian depletion induced by chemotherapy for the treatment of malignancies.20
Serum levels of estradiol fluctuate throughout the estrous cycle and as such, estradiol is not a reliable predictor of reproductive function.19 Estradiol is produced by the granulosa and theca cells of the developing follicle. The production of oestradiol is under the control of gonadotropins from the anterior pituitary. Estradiol regulates female reproductive functions and it is diminished with aging and depletion of follicles.10 HAART has been shown to counteract the contraceptive benefits of the estrogen-containing oral contraceptive pill and invariably leads to a higher contraceptive failure rate.19
In this study, there was an increased average level of estradiol in the HAART study groups, when compared to the control group. This change was not statistically significant. However, some workers have noted elevated levels of estradiol in males who are being treated with antiretroviral drugs; this elevation leads to sexual dysfunction in this male.21 There is a need to further define if this finding applies to HIV-positive women on HAART.
Reduced levels of estrogen obliterate the negative feedback of estrogen on FSH. This leads to a marked elevation of FSH. Elevation of FSH is used as a marker of ovarian function, although it is not as reliable and accurate as the use of AMH and antral follicular count. Concerns have been raised about the age of menopause of women being managed for HIV. It is thought that women who are being managed for HIV will get to menopause earlier than their HIV-negative counterparts.22 FSH levels in these individuals have been used as a marker for ovarian function in this group of individuals, although challenges do exist with its usage, due to fluctuation through the menstrual cycle.23