Efavirenz Based First Line Anti-Retroviral Drugs Disrupts Follicular Development-Evidence From Animal And Human Studies


 INTRODUCTION: Interventions to eliminate mother to child transmission of HIV has led to very low vertical transmission rate, however there are genuine concerns on long term effect of anti-retroviral drugs on future reproductive functions of females. There is currently paucity of information regarding the effect of anti-retroviral agents such as Tenofovir, Efavirenz and Lamivudine on the ovarian reserve of patients receiving anti-retroviral drugs. METHODOLOGY: 1. ANIMAL STUDY: A total of 25 female Wistar rats, weighing between 150g to 170g were obtained and kept in the experimental animal house of the Nigerian Institute of Medical Research (NIMR) in Lagos, Nigeria. They were kept in clean cages at a temperature of 28 o C HUMAN STUDY: Women within the reproductive age range of 15-45 years were grouped into A, B and C categories. Group A was made up of HIV positive patients already on efavirenz based anti-retroviral therapy. Group B were HIV positive patients who were naïve to antiretroviral drugs at the time of recruitment. Group C participants were HIV negative women within the reproductive age range not being managed for Infertility. RESULTS: ANIMAL STUDY : The mean count of antral follicles was significantly lower in the group that received efavirenz (p=0.00). The corpus luteal count was significantly higher in the control group than study groups (p=0.033) HUMAN STUDY: Average value of FSH is significantly higher in the HIV positive group who had antiretroviral medications when compared to HIV negative group. (P=0.039).Antral follicular count was significantly reduced in the group that received anti retrovirals (ARV). P=0.009 CONCLUSION: Evidence from this animal and human study, tend to suggest that efavirenz based antiretroviral medication disrupts follicular development.

to eliminate mother to child transmission of HIV has led to very low vertical transmission rate (Eunice, 2012).
There are however challenges with long-term usage of the anti-retroviral drugs as side effects of these drugs have become issues of concern. For instance Nucleoside reverse transcriptase inhibitors have been implicated in mithochondrial damage and haematological toxicity leading to anaemia (Dalakas, 1990;Arnaudo, 1991;Brinkman, 2000;Taedor, 2004)..
Adverse effects related to the central nervous system have been implicated in nonnucleoside reverse transcriptase inhibitors (Marzolini, 2001;Jeromea, 2001).There is some evidence to suggest that some of the side effects can affect the reproductive efficiency of the patients as evidence abound that some anti-retroviral drugs can affect sperm motility in some males (Van Leewen, 2008). There is also suggestion that antiretroviral drugs can lead to testicular atrophy (Mhawech, 2001). This evidence has not been clearly established especially for the female gametes. Now that HIV positive individuals can live relatively normal lives, when managed with highly active anti-retroviral drugs, there is therefore the desire for most of these patients to consider raising children and the integrity of their reproductive potential becomes an issue of concern.
There is currently paucity of information regarding the effect of anti-retroviral agents such as Tenofovir, Efavirenz and Lamivudine on the ovarian reserve of patients receiving anti-retroviral drugs.
There are however no studies in animal models or humans in our environment to evaluate the effect of anti-retroviral drugs on the reproductive functions of female patients receiving highly active antiretroviral drugs.
This therefore is the rational for the conduct of this study OBJECTIVE OF THE STUDY:

1.
To ascertain the effect of Lamivudine, Tenofovir and Efavirenz on the follicular development of wistar rats 2.
To determine the antral follicular count of HIV negative women within the reproductive age range.
1.1 ml of 0.04 mg/ml solution was given to a 150 g rat 3.3.3 PROCEDURE FOR SACRIFICING: The rats were sacrificed during the oestrous phase of the cycle. The rats were inactivated by cervical dislocation. The skin and the subcutaneous tissue including the peritoneum were exposed to the abdomen. Blood samples were obtained from the heart. Some of the organs obtained include the ovaries, the oviducts and the uterus. These organs were preserved in saline solution initially and subsequently transferred to formalin solution.

HISTOLOGY AND FOLLICULAR COUNT:
The ovarian tissue obtained was be fixed in 10% formalin and thereafter dehydrated in a stepwise manner by the use of ethanol. After this process, it was cleared in Xylene and then embedded in a wax of paraffin. A section of the ovarian tissue embedded in paraffin was stained with Eosin and

ULTRASOUND PROCEDURE FOR ANTRAL FOLLICULAR COUNT:
Antral follicular count is performed for female humans during the follicular phase of the menstrual cycle prior to ovulation. The procedure is performed more specifically during the early follicular phase after the menstrual period.
The preferred method for the determination of the antral follicular count is via a vaginal ultrasound scan at a frequency of 6-8 mega hertz. The Mindray M7 ultrasound machine was used for this procedure. The frequency of the vaginal transducer to be used is 7megahertz.
The patients were adequately counselled for the procedure and an additional verbal consent obtained after the signed informed consent used to recruit patients for the study. A female chaperone was present all through the procedure. The transducer was covered with a protective sheath before a gel was applied to it. This was done to eliminate the risk of infection. The aim of the gel is to improve the

INFORMED CONSENT:
A written informed consent was obtained from the patients. Average value of FSH is significantly higher in the HIV positive group who had antiretroviral medications when compared to HIV negative group. P=0.039

ANTRAL FOLLICULAR COUNT ACROSS THE THREE COHORTS
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 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,

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. 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, 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

Conclusions
This study has shown that long term usage of efavirenz based anti-retroviral medications is associated with compromised follicular development and dysfunctional reproductive hormone profile.
The implication of this finding as it relates to use of efavirennz based anti-retroviral medications in the management of women in the reproductive age range is far reaching. There is therefore an urgent need to review the current regimen in use for the management of women within the reproductive age range who are being managed with efavirenz based combination