J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 99-105. https://doi.org/10.47277/JIRB/8(4)/99  
Antifertility Potential of Isoniazid and Rifampicin  
in Adult Female Wistar Rats  
Vitalis Chukwuma Ezeuko*, Jacob Ehiagwina Ataman  
Department of Anatomy, School of Basic Medical Sciences, College of Medical Sciences, University of Benin, Benin City, Edo State, Nigeria  
Received: 06/09/2020  
Accepted: 25/11/2020  
Published: 20/12/2020  
Abstract  
The aim of this study was to investigate the effects of INH and RIF on the reproductive functions of adult female Wistar rats. Twenty-  
eight adult female Wistar rats weighing between 160 g and 170 g were divided into four groups. Group A served as the control. Group  
B was given 5 mg/kg body weight of INH. Group C animals was given 10 mg/kg body weight of RIF. Group D animals received a  
combination of 5 mg/kg body weight of INH and 10 mg/kg body weight of RIF. Duration of treatment was 90 days and via oral route.  
Histological findings showed that INH and RIF caused histopathologic changes in the ovary and uterus. Compared with the control, the  
ovarian SOD was significantly elevated in all the treated groups. Ovarian MDA, CAT and GPx activities were significantly elevated in  
INH-only treated group. FSH was significantly elevated in RIF-only and INH and RIF co-treated groups. LH was significantly elevated  
in all the groups. Progesterone level was significantly reduced in INH-only and INH and RIF co-treated groups. Estradiol level was  
significantly elevated in RIF-only and INH and RIF co-treated groups. Testosterone level was significantly elevated in INH-only treated  
group. Prolactin level was significantly reduced in all the groups while testosterone:estradiol ratio was significantly elevated in INH-  
only treated group but significantly reduced in INH and RIF co-treated group. In conclusion, administration of INH and RIF caused  
toxicity in the female reproductive system.  
Keywords: Antitubercular agents, Rifampin, Isoniazid, Female, Reproductive health  
Introduction  
The broad viewpoints of Medical practitioners on  
tuberculosis (TB) in pregnancy reflects the Public Health  
significance of the condition, with the concern being two ways.  
One concern is about the effect of TB on pregnancy and the  
pattern of growth of the newborn, while the other is the effect  
of pregnancy on the progression of TB. TB not only accounts  
for a significant proportion of the global burden of disease, it is  
also a significant contributor to maternal mortality, with the  
disease being among the three leading causes of death among  
women aged 15-45 years (1).  
instance, rifampicin is said to belong to category C drugs and  
recommended for use even in pregnancy as there has not been  
any claim of its teratogenic effects (19-21). However, it has  
been shown that neonates born to mothers treated with  
isoniazid exhibit hemorrhage in the postpartum period (22).  
This study is intended to investigate and broaden the  
perspective of the effects anti-TB drugs on female reproductive  
parameters.  
Materials and Method  
The combination of rifampicin and isoniazid along with  
ethambutol and pyrazinamide has been adjudged best for  
efficacy and tolerability amongst the available TB drugs and is,  
therefore, the mainstay “first line” therapy (2, 3). There are  
historical and clinical proofs that these first-line anti-  
tuberculosis agents are the most potent oral anti-tuberculous  
medications (4-6). In addition, in-vitro and in-vivo clinical data  
support the use of such individual agents (7, 8). This may be  
because the combination has been found to be a beneficial and  
cost-effective treatment for TB (9), but, this is not without some  
systemic toxicity from the results from human data.  
Since TB affects people in their productive and  
reproductive age group, there have been recent suggestions that  
anti-tuberculosis agents could produce adverse effects on  
reproductive health system (10-13). Available data on adverse  
reactions of antituberculous drugs in human and animal  
experiments particularly with respect to reproduction are  
limited (14). Most of the few available studies on effects of  
anti-tuberculosis agents on reproduction and/or fertility had  
focused on males (15, 16) but with scanty literature on female  
fertility (17, 18).  
Drugs  
Rifampicin capsules (manufactured by Bond Chemicals  
Limited, Adesakin Layout, Awe, Oyo State, Nigeria) and  
isoniazid tablets (manufactured by Mancare Pharmaceutical  
Pvt Limited, Vasai West, Mumbai, India) were purchased from  
Flowell Pharmacy, 39 Ugbowo-Lagos Road, Benin City, Edo  
State, Nigeria.  
Animals  
Wistar rats used for the study were bred at the Animal  
House, Department of Anatomy, School of Basic Medical  
Sciences, College of Medical Sciences, University of Benin,  
Benin City Edo State Nigeria. They were kept in polypropylene  
cages under room temperature, with 12-hour light and 12-hour  
dark cycle photoperiodicity. They were allowed to acclimatize  
for two weeks before the commencement of the experiment.  
The animals were fed with pelleted feed (manufactured by  
Grand Cereals and Oil Mills Ltd, Bukuru, Jos, Nigeria) and  
clean tap water ad libitum. They were weighed daily before the  
commencement and throughout the duration of the experiment.  
Protocols for these experiments were in accordance with the  
guide for the care and use of laboratory animals (23).  
There is need to continuously review most drugs consumed  
by mankind including those generally considered as safe. For  
*
Corresponding author: Vitalis Chukwuma Ezeuko, Department of Anatomy, School of Basic Medical Sciences, College of Medical Sciences,  
University of Benin, Benin City, Edo State, Nigeria. Email: chukwuma.ezeuko@uniben.edu  
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J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 99-105. https://doi.org/10.47277/JIRB/8(4)/99  
The research proposal was approved by the Research and  
with the control group using students’ t-test. Differences in  
means were considered significant at 95 % confidence level  
(that is when probability was less than 0.05 {P < 0.05})  
Ethics Committee, College of Medical Sciences, University of  
Benin, Benin City Edo State Nigeria, with REC approval  
number CMS/REC/2018/034.  
Results  
Research design  
Table 1 shows the initial and final body weight, body  
weight changes, ovarian weight, relative ovarian weight,  
uterine horn weight and relative uterine weight of all the  
experimental groups. From the Table, there was no significant  
difference (P>0.05) in the initial body weight (body weight  
before the commencement of the experiment) in all the  
experimental groups. Comparison between initial and final  
body weights in each group showed that there was significant  
increase (P<0.05) in body weight in all the groups. There was  
no significant difference (P>0.05) in the mean weight changes  
in all the experimental groups, compared with the control  
A total of twenty-eight (28) adult female Wistar rats  
weighing between 160 g and 170 g were used for this study.  
They were divided into four groups (A, B, C and D) of seven  
rats per group. Group A served as the control group  
administered with only water orally and feeds for ninety days.  
Group  
B animals, in addition to normal feeds, were  
administered 5 mg/kg body weight of isoniazid orally for ninety  
days. Group C animals, in addition to feed, were administered  
1
0 mg/kg body weight of rifampicin orally for ninety days.  
Group D animals, in addition to normal feed, were orally  
administered 5 mg/kg body weight of isoniazid and 10 mg/kg  
body weight of rifampicin for ninety days.  
The dosages of rifampicin and isoniazid used in this study  
are consistent with the recommended daily dosages for humans  
which are 5 mg/kg body weight/day for isoniazid and 10 mg/kg  
body weight/day for rifampicin (17, 27).  
After ninety days of administration, the animals were  
humanely sacrificed under ketamine anesthesia. The ventral  
abdominal wall was opened and blood samples were collected  
via the abdominal aorta for hormone analyses while the ovaries  
and uterus were harvested for oxidative stress analyses and  
histological studies.  
The hormones were assayed according to the principle of  
competitive enzyme immunoassay using AccuBind ELISA  
Microwells manufactured by Monobind Inc. Lake Forest, CA  
(
Table 1).  
The ovarian weight was significantly lower in the isoniazid  
treated group (P<0.05) and in the rifampicin treated group  
P<0.05), compared with the control. However, there was no  
(
significant difference (P>0.05) in the ovarian weight between  
the rifampicin-isoniazid co-treated group and the control. There  
was no significant difference (P>0.05) in relative ovarian  
weight when all the experimental groups were compared with  
the control (Table 1).  
There was no significant difference in the uterine horn  
weight between the isoniazid treated group (P>0.05) and the  
control but the uterine horn weight was significantly lower in  
the rifampicin treated group (P = 0.022) and isoniazid-  
rifampicin co-treated group (P = 0.043), compared with the  
control (Table 1).  
There was no significant difference (P>0.05) in the relative  
uterine weight between the isoniazid treated group and the  
control. However, relative uterine weight was significantly  
lower in the rifampicin treated (P<0.05) and isoniazid-  
rifampicin co-treated groups (P<0.05), compared with the  
control (Table 1).  
Table 2 shows the mean ovarian oxidative stress parameters  
of all the experimental groups. From the Table, there was  
significant increase (P<0.05) in superoxide dismutase activity  
in all the experimental groups compared with the control. There  
was significant increase (P<0.05) in malondialdehyde activity  
in isoniazid treated group, compared with the control but there  
was no significant difference in malondialdehyde activity of the  
rifampicin treated group (P>0.05) and isoniazid-rifampicin co-  
treated group (P>0.05), compared with the control. There was  
a significant increase (P<0.05) in catalase activity in isoniazid  
treated group, compared with the control but there was no  
significant difference in catalase activity of the rifampicin  
treated group (P>0.05) and isoniazid-rifampicin co-treated  
group (P>0.05), compared with the control. There was  
significant increase (P<0.05) in glutathione peroxidase activity  
in isoniazid treated group, compared with the control but there  
was no significant difference in glutathione peroxidase activity  
of the rifampicin treated group (P>0.05) and isoniazid and  
rifampicin co-treated group (P>0.05), compared with the  
control.  
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2630, USA). The procedure for the enzyme immunoassay was  
followed according to the manufacturer’s guidelines.  
The ovaries were, immediately after harvesting, blotted free  
of blood and weighed immediately using an electronic  
weighing balance (manufactured by Kern & Sohn GmbH, D-  
7
2336 Balingen, Germany) calibrated in milligram and  
recorded to the nearest two decimal places. The relative ovarian  
weight was evaluated as the percentage of absolute ovarian  
weight divided by body weight while the relative uterine weight  
was evaluated as the percentage of absolute uterine horn weight  
divided by body weight.  
One of the ovaries was then washed twice in cold phosphate  
buffered saline (PBS) after which it was homogenized using  
acid-washed sand and PBS in porcelain mortar and pestle. The  
tissue homogenate was centrifuged at 10000 rpm for 10  
o
minutes at 4 C. The supernatant was immediately processed for  
analysis of endogenous antioxidants. Superoxide dismutase  
activity was determined according the method of Misra and  
Fridovich (24). Catalase activity was determined according the  
method of Cohen et al., (25). Glutathione peroxidase activity  
was determined according the method of Nyman (26).  
The second ovary and uterus were preserved in 10 %  
phosphate buffered formalin for histopathology. The tissues  
were processed via paraffin wax embedding method of Drury  
and Wallington (28). Procedures of Haematoxylin and Eosin  
adopted on the sections were as described by Drury and  
Wallington (28) and Scheehan and Hrapchak (29).  
Table 3 shows the results of treatments on the hormonal  
profile of all the experimental groups. From the Table, there  
was no significant difference (P>0.05) in follicle stimulating  
hormone level between the isoniazid treated group and the  
control group but the follicle stimulating hormone level was  
significantly lower in the rifampicin treated group (P<0.05) and  
in the isoniazid-rifampicin co-treated group (P<0.05),  
compared with the control. The luteinizing hormone levels  
Statistical Analysis  
The data were analyzed using IBM Statistical Package for  
Social Sciences, Version 23 (manufactured by International  
Business Corporations {IBM}; released in 2015). Results were  
presented as mean + standard error of mean (mean + SEM).  
The parameters for all the experimental groups were compared  
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J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 99-105. https://doi.org/10.47277/JIRB/8(4)/99  
were significantly higher (P<0.05) in all the experimental  
ovary is enveloped by germinal epithelium (GE). The  
photomicrograph of the ovary of rats treated with isoniazid  
only (Figure 1, upper right) shows ovarian atrophy in the cortex  
with absence of interstitial glands and non-remarkable  
medulla, reduced number and growth of developing follicles  
groups compared with the control. The progesterone levels  
were significantly lower in isoniazid treated group (P<0.05)  
and in isoniazid-rifampicin co-treated group (P<0.05),  
compared with the control but there was no significant  
difference (P>0.05) in progesterone level between the  
rifampicin treated group and the control. The estradiol levels  
were significantly higher in rifampicin treated group (P<0.05)  
and in isoniazid and rifampicin co-treated group (P<0.05),  
compared with the control but there was no significant  
difference (P>0.05) in estradiol level between the isoniazid  
treated group and the control. The testosterone level was  
significantly higher (P<0.05) in isoniazid treated group,  
compared with the control but there were no significant  
differences in testosterone level between the rifampicin treated  
group and the control (P>0.05) and between the isoniazid and  
rifampicin co-treated group and the control (P>0.05). The  
prolactin levels were significantly lower (P<0.05) in all the  
experimental groups compared with the control. The  
testosterone:estradiol ratio was significantly higher (P<0.05) in  
isoniazid treated group when compared with the control group  
and significantly lower (P<0.05) in isoniazid and rifampicin co-  
treated group, compared with the control but there were no  
significant differences in testosterone:estradiol ratio between  
the rifampicin treated group and the control group(P>0.05).  
The photomicrograph of the ovary of the control group  
o
(1 ) compared to the control, atretic tertiary follicle (aTF) and  
degenerating corpora lutea (dCL). There is presence of  
interstitial stromal cell hyperplasia arranged in variably sized  
island and clusters (IC) and follicular cysts (FC). The  
photomicrograph of the ovary of rats treated with rifampicin  
only (Figure 1, lower left) shows atretic tertiary follicle (aTF),  
degenerating corpora lutea (dCL); follicular cysts (FC) and  
generalized haemorrhagic inflammation of the ovary (Hg). The  
photomicrographs of the ovary of rats treated with isoniazid  
and rifampicin (Figure 1, lower right) shows reduced number  
of developing follicles, presence of follicular cysts (FC) and  
petechial hemorrhage (Hg) in the ovarian stroma.  
The photomicrograph of the uterine horn of the control  
group (Figure 2, upper left) shows normal histological features  
with a patent endometrial canal (ec), inner endometrium (En),  
middle myometrium (My) and outer perimetrium (Pe). The  
photomicrograph of the uterine horn of the isoniazid-only-  
treated group (Figure 2, upper right) showed cystically dilated  
glands (cg) in the endometrium. The photomicrograph of the  
uterine horn of the rifampicin-only-treated group (Figure 2,  
lower left) shows mildly congested blood vessels (BV) in the  
lamina propria (LP) and myometrium. The photomicrograph of  
the uterine horn of the isoniazid and rifampicin co-treated  
group (Figure 2, lower right) shows severe endometrial  
hyperplasia (EH) obliterating the uterine lumen.  
(
Figure 1, upper left) shows normal histological features of the  
o
o
ovary with primary follicles (1 ), secondary follicles (2 ), and  
corpora lutea (CL) present in the cortex; blood vessels (BV)  
and interstitial glands (IG) are present in the medulla (M); the  
Table 1: Body weight, ovarian weight and uterine horn weight in animals of different groups  
Relative  
Relative  
uterine  
weight  
0.202+0.05  
0.201+0.03  
0.114+0.01*  
0.148+0.02*  
Weight  
Ovarian  
weight  
Uterine horn  
weight  
Initial weight  
Final weight  
ovarian  
weight  
0.041+0.005  
0.030+0.005  
0.029+0.004  
0.038+0.004  
difference  
a
b
Control  
168.00+3.70  
213.86+13.89  
45.86+10.57  
44.57+2.65  
56.57+21.15  
55.00+5.22  
0.086+0.008  
0.064+0.006*  
0.061+0.004*  
0.087+0.011*  
0.552+0.087  
0.417+0.059  
0.162+0.018*  
0.336+0.028*  
a
b
Isoniazid only  
Rifampicin only  
Isoniazid +rifampicin  
166.86+3.75  
211.43+4.26  
a
b
165.43+3.19  
222.00+24.30  
a
b
224.14+ 8.03  
169.14+4.43  
Comparing initial and final weight within a group. Data are presented as mean + SEM. Unlike superscript means significant difference (P < 0.05).  
Comparing other parameters across the groups, ‘*’ means significant difference (P < 0.05).  
Table 2: Ovarian Oxidative Stress in animals of different groups  
GPX  
Catalase  
0.060+0.011  
0.093+0.011*  
0.076+0.010  
0.069+0.021  
Malondialdehyde  
0.000123+0.00002  
0.000195+0.00002*  
0.000128+0.00002  
0.000115+0.00002  
SOD  
a
Control  
0.281+0.070  
0.440+0.038  
0.731+0.156*  
0.611+0.040*  
0.705+0.057*  
Isoniazid only  
Rifampicin only  
Isoniazid + rifampicin  
0.577+0.336*  
0.336+0.079  
0.423+0.076  
Comparing the parameters across the groups. Data are presented as mean + SEM. ‘*’ means significant difference (P < 0.05).  
Table 3: Hormone profile in animals of different groups  
FSH  
LH  
PRG  
Estradiol  
14.80+2.36  
11.26+5.70  
20.28+0.76  
26.34+2.42*  
Testosterone  
0.13+0.02  
PRL  
2 2  
T : E ratio  
Control  
0.78+0.11  
0.88+0.17  
0.44+0.05*  
0.42+0.04*  
0.13+0.03  
0.42+0.04*  
0.84+0.05*  
1.92+0.31*  
17.30+1.08  
8.82+0.62*  
14.62+1.68  
3.42+0.18*  
0.48+0.02  
0.30+0.03*  
0.16+0.04*  
0.22+0.02*  
0.009+0.02  
0.084+0.043*  
0.007+0.001  
0.005+0.001*  
Isoniazid only  
Rifampicin only  
Isoniazid + rifampicin  
1.08+0.032*  
0.14+0.024  
0.12+0.020  
Comparing the parameters across the groups. Data are presented as mean + SEM. ‘*’ means significant difference (P < 0.05).  
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