ANIMAL STUDY
Folliculogenesis is a key component of reproductive functions in both lower animals and human subjects. Folliculogenesis is the process that leads to the formation of a preovulatory or dominant follicle that houses the oocyte. Folliculogenesis involves the primordial follicle maturing to become the primary follicle, the secondary or pre-antral follicle and then the tertiary or antral follicle.(White and Porterfield, 2012). The antral follicle eventually evolves to become the pre-ovulatory follicle . During folliculogenesis, a number of follicles are destroyed and end up as atretic follicle. (White and Porterfield , 2012)
The rat model is ideal for the study of folliculogenesis due to the relatively short cycle length averaging 4.8 days (Andrew and Ojeda, 1981). Follicular development is under the control of both hormornal and growth factors. (Fortune 2003).
Findings from this study revealed a reduction in the average number of follicles across various stages of follicular development in the study groups when compared to control. This reduction in the average number of follicles showed statistical significance (P=0.00). The reduction in the number of follicles was more marked in the study group that received efavirenz when compared to the study group that received, tenofovir, lamivudine and a combination of all three drugs. The affectation was more with follicles at the antral stage of development. The reduction of follicles in the group that received efavirenz was not seen in the other study groups receiving other anti-retroviral medications-tenofovir and lamivudine. Efavirenz based combination is in common usage as first line anti-retroviral combination in HIV positive women within the reproductive age range.
A decrease in the number of follicles has been observed in human females as they age. Smoking is also associated with a decrease in the number of follicles. Decrease in follicles has also been observed in individuals who are receiving cytotoxic medications as well as those who are exposed to radiotherapy. (Peck, 2016).
A decrease follicular count was however documented in female wistar rats exposed to Heracleum persicum (H. persicum). (Hemati, 2012 ). This is a herbal medicinal plant famously used in Iran and Asiantic nations of the world for a variety of medicinal purposes. This plant has been used in some traditional settings as a form of contraception.
The average level of Anti-mullerian hormone in this study was lower in the study group that received anti-retroviral therapy when compared with the control group . The average AMH in the cohort of rats that received the combined anti-retroviral therapy and efavirenz alone was lower than the control. The difference was however not statistically significant.(p=0.180). AMH (Anti-mullerian hormone ) is a marker for reproductive potential and ovarian function. Levels are very low in neonates and early childhood but begin to rise during puberty. As reproductive capacity declines with age due to depleting ovarian reserve, there is a decrease in anti-mullerian hormone. As the menopause is being approached, anti-mullerian hormone diminishes. AMH values thus correlates 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. (Kevenaar, 2006). AMH is used for the estimation of ovarian reserve and a prediction of the fertility potential in females. The correlation of AMH with antral follicular count is due to the fact that AMH is secreted by the granulosa cells of the developing follicle. Changes in the level of AMH are known to occur relatively early as it relates to ovarian aging. Changes in AMH IS known to predate changes in FSH levels and inhibin B.( Nathalie and Nathalie 2004). Anti-mullerian hormone is thus a better predictor of ovarian reserve than Inhibin B and FSH. It is also a more reliable marker of ovarian reserve because it does not fluctuate through the menstrual cycle .(Nathalie and Nathalie 2004). In humans, normal levels of AMH is 1.5-4.0ng/m/.This hormone is also relevant in-utero for inhibiting mullerian growth and the the evolution of the fetus to the male sex. (White and Poterfield ,2012). Recent studies tend to suggest that AMH could be protective against ovarian depletion induced by chemotherapy for the treatment of malignancies. (Motohiro, 2016).
Changes in the level of AMH have been shown to influence litter size in mice. Mclennan I.S. and co-workers demonstrated that mice with either elevated or diminished levels of anti-mullerian hormone have reduced litter size. (Mclennan, 2017).
Serum levels of oestradiol fluctuate throughout the oestrous cycle and as such oestradiol is not a reliable predictor of reproductive functions. (Nathalie and Nathalie , 2004). Oestradiol is produced by the granulosa and theca cells of the developing follicle. The production of oestradiol is under the control of gonoadotropins from the anterior pituitary. Oestradiol regulate female reproductive functions and it is diminished with ageing and depletion of follicles.(White and Poterfield, 2012)
In this study, there was a diminished average level of oestradiol in the study group when compared to the study groups. This elevation was however not statistically significant. Some workers have however noted elevated levels of oestradiol in males who are being treated with anti-retroviral drugs, this elevation lead to sexual dysfunction in these males. (Lamba, 2004). There is need to elucidate through research if this finding applies to HIV positive women on highly active anti-retroviral therapy.
Anti-retroviral drugs have been known to counteract the contraceptive benefits of oestrogen containing oral contraceptive pill and invariably leads to higher contraceptive failure rate. (Anderson, 2007)
THE EFFECT OF ANTI-RETROVIRAL DRUGS ON THE SERUM LEVEL OF FSH AND LH
FSH AND LH are both referred to as gonadotropins. These hormones are products of the anterior pituitary. LH and FSH play crucial roles in the development of the follicles and other reproductive functions They are trophic hormones and as such they are known to stimulate the gonads (White and Poterfield , 2012).
In this 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 significant degree of variation. FSH level in the group that received TDF was reduced when compared to the control group. FSH level was however markedly elevated in the group that received FDC when compared with the control group, this difference was statistically significant (p=0.05). In other study groups that received efavirenz , FSH was marginally elevated when compared to the control group. FSH was however markedly elevated in the lamivudine group when compared against control, but this difference was not statistically significant (p=0.08). FSH level is known to correlate with ovarian failure and serum level of oestrogen. This relationship is inverse as depletion of ovarian follicles, leads to reduced levels of oestrogen. The reduced levels of oestrogen obliterates the negative feedback of oestrogen on FSH. This leads to 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 the menopause earlier than their HIV negative counterparts. (Fan,2008).There is however no clear evidence to suggest if this is due to the effect of the disease or the medications.FSH levels in these individuals have been used as a marker for ovarian function in these group of individuals, although challenges do exist with its usage, due to fluctuation through the menstrual cycle.(Kahwati,2005).
HUMAN STUDY
The study involved women within the reproductive age range. The average age of the HIV negative patients was 32 years , while that of the HIV positive patients without drug was 33.40 years and that of the HIV positive patients with drug was 37.58 This showed that the human research subjects were well matched for age, this minimizing the risk of bias. Moreover, individuals within this age category are still actively involved with reproductive processes.
Fertility and reproductive potential has been known to decline with advancing age. (Liu , 2012). The table also showed that most of the participants across the three study groups did not smoke nor consume alcohol. Smoking and alcohol consumption is a cofounder in this study as binge drinking has been shown to be associated with premature ovarian failure and depletion of ovarian reserve.(Hawkins, 2016)
Female smoking has been shown to be associated with depletion in ovarian reserve. (Firns, 2015).
Antral Follicular Count (AFC) is a marker for ovarian reserve and a reflection of the fertility potential of human females. It is the gold standard for the measurement of ovarian reserve of human females. (ACOG,2015). It can be used in isolation in the determination of patients who will require egg donation in preparation for Invitro fertilization using donor egg. (ACOG, 2015).
AFC measurement in this study was done with the aid of a four dimensional colour Doppler ultrasound machine with a transvaginal transducer. Colour Doppler ultrasound is superior to plain ultrasound machine because they can discriminate follicles from blood vessels. The study revealed that AFC was significantly lower in HIV positive individuals receiving anti-retroviral therapy when compared to the participants in the other cohorts (p=0.009). Antral follicular count values have been shown to be a better predictor of hyperstimulation in IVF cycles when compared to AMH. (Christianson M.S., 2016).
This reduction in AFC noticed in the human population was also documented in the rat model used for this study. The reduction in AFC in the human population was also reflected in the findings obtained from estimation of the FSH level of the human research subjects used for the study as discussed below.
AFC estimation is done during the follicular phase of the menstrual cycle, usually between days 7-10 as this is the period of the cycle were follicular recruitment occurs. Values above 8 are considered normal. The normal range is between 8-16 antral follicles. Patients with Polysystic ovarian syndrome (PCOS) tend to have high AFC . AFC is usually over 18. .
HIV has been shown by some studies to negatively impact the values of AFC.(Ohl .J, 2010). The impact of anti-retroviral medications on AFC is however not clear.
The average CD4 values across the study population who are HIV positive was 725 and the average viral load was 20 copies per ml. This finding shows that human participants used for the study, were immunologically and virologically stable, hence the influence of the pathology of the disease condition cannot be used as a cofounder influencing the findings above. Patients with viral load of 20 copies/ ml are classified as undetectable viraemia. These patients are therefore in a good state of health.
HORMONE PROFILE ACROSS THE THREE GROUPS
In these cohorts of human participants, used for the conduct of the study, serum levels of FSH, LH, and Oestradiol were evaluated during the follicular phase. Serum levels of these hormones are known to fluctuate during the menstrual cycle, hence values used in the evaluation of patients are best done during the follicular phase of the cycle, when follicular recruitment is taken place. (Fritz and Speroff, 2011). The mean FSH value for the research participants who are positive and on drug was higher than the other two cohorts. Average value of FSH is significantly higher in the HIV positive group who had anti-retroviral medications when compared to HIV negative group. P=0.039. On the contrary, there was no statistical significant difference between the average FSH levels in HIV positive individuals not on drug and HIV negative individuals when compared .(p=0.222). One can possibly infer from this finding that HIV positivity did not significantly lead to a higher than normal FSH values in these individuals when compared to their HIV negative counterparts. However HIV individuals who have been on long term usage of efavirenz based combination tend to have higher than normal FSH values when compared to individuals in the other two cohorts.
A Normal FSH value in reproductive aged women during the follicular phase is 3.77.9miu/ml. (Sofronescu G.A., and Wheeler M.T.,2015). FSH value is a predictor of ovarian reserve and should directly correlate with values of antral follicular count. The average FSH value in patients who are HIV positive and on drug was higher than the range for normal. This is also true for patients who are HIV positive and not on drug, but the patients who are HIV positive and on drugs had a the worst FSH value. Values above 30.6miu/ml are considered as menopausal. (Sofronescu G.A., and Wheeler M.T., 2015). It should be noted however that FSH value is a late predictor of ovarian reserve as AMH and antral follicular count are early and more accurate predictors of ovarian reserve. (Nathalie and Nathalie, 2004). Santoro and co-workers who studied the factors affecting reproductive hormones in HIV positive substance using women within the middle age range came up with the conclusion that use of anti-retroviral drugs in these cohort of patients was associated with higher FSH , LH and Oestradiol when compared with controls. (Santoro. N., 2007).
Estimation of serum levels of oestradiol for reproductive functions is usually done during the follicular phase of the cycle during recruitment of the follicles. Normal range for oestradiol in human females is 27-123pg/ml.(Sofronescu A.G. and Staros B.E., 2015) . In this study, the average serum level of oestradiol was lower than the average serum level of oestradiol in the other two cohorts. The average serum level of oestradiol was 36.00pg/ml for HIV positive on drugs while that of HIV positive not on drug was 52.50pg/m/. The average serum level of oestradiol for respondents who are HIV negative was 38.20ng/m/. This differences were however not statistically significant. A low level of oestradiol is present in premature ovarian failure and the menopause. Oestradiol levels tend to exhibit an inverse relationship with levels of Follicle stimulating hormone and a direct relationship with levels of AMH.(Fritz and
Speroff,2011). This pattern is not always the case as serum levels of both oestradiol and FSH are late and less than accurate predictors of ovarian reserve. (Nathalie and Nathalie , 2004). Oestradiol levels below 40pg/ml are reflective of a value close to that in the menopausal range.
The normal range of LH during the follicular phase is 1-18miu/ml. (Charbek.E. and Wheeler M.T, 2015). LH is not a reliable predictor of ovarian reserve and reproductive potential because of its fluctuation over the cycle duration and also because of the broad range of value. LH is however useful when interpreted in conjunction with FSH when making a diagnosis of Polycystic ovarian syndrome. In this regard LH/FSH ratio becomes relevant. The normal value is 1:1 but in patients with PCOS, the ratio becomes 2:1 or 3:1 due to the elevated values of LH. (Fritz and Speroff, 2011) .
In this study, average LH value was more elevated in the HIV positive group than the HIV negative group. The elevation was more marked in the HIV positive group on drug than the HIV positive group not on drug. This difference was statistically significant(p=0.014). Elevated LH levels when used in conjuction with FSH levels suggest anovulation . (Santoro N., 2007)
RELATING THE ANIMAL STUDY AND THE HUMAN STUDY
The rat model is a good model for the study of reproductive functions in higher mammals because of the relatively short cycle length and it allows for in-depth study of parts of the reproductive system of higher animals through relative ease of obtaining tissue from the reproductive system for study.
A significant reduction in the antral follicles of the wistar rats that received the medication was also observed (p=0.00). This was also mirrored in the findings from the human study that revealed a reduction in the mean level of antral follicular count in the HIV positive patients on medication (p=0.009)
Surrogate markers of ovarian reserve such as AMH and FSH values were consistently in favour of diminished ovarian reserve in the group of rats that had the anti-retroviral medication. This same effect was demonstrated in the human population as serum levels of FSH showed pattern consistent with relatively diminished ovarian reserve in patients receiving anti-retroviral therapy when compared to age matched controls who are HIV positive but not receiving anti-retroviral therapy