Pharmacological effects of low- dose of aspirin on ovulation rate in mature cycling female mice
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Middle East Fertility Society Journal Vol. 12, No. 1, 2007
Copyright © Middle East Fertility Society
Pharmacological effects of low- dose of aspirin on
ovulation rate in mature cycling female mice
Adnan S. Al-Janabi, B.V.M.S., Ph. D.
Ahmad M. Al-Zohyri, B.V.M.S., Ph. D.
Fouad K. Al-Rubayai, M.B.Ch.B , M.Sc.
Institute of the Embryo Research and Infertility Treatment, College of Medicine, University of Baghdad, IRAQ.
ABSTRACT
Objective: To determine the effects of low-dose of aspirin on ovarian response, uterine and ovarian blood flow supply,
and ovulation rate in mature cycling female mice.
Design: Mature cycling female mice treated with aspirin on the first day of proestrous phase.
Setting: Institute of the Embryo Research and Infertility Treatment- University of Baghdad.
Materials and methods: Mature cycling female mice treated subcutaneously twice/day with Aspirin (7.5mg/Kg.b.w)on
the first day of proestrous phase. In the treated six groups (6 mice/ group) underwent subcutaneous administration of
aspirin at a dose level of (7.5mg/Kg b.w) twice daily at proestrous phase of the estrous cycle. In the control six
group (6mice/ group) underwent subcutaneous administration of a placebo (distilled water) twice daily at proestrous
phase of the estrous cycle
Main outcome measure(s): Uterine and ovarian morphological changes, uterine and ovarian weight changes, number
of ova and serum (FSH, LH and E2) levels.
Results: There were statistically significant differences between the treated group and the control group, respectively,
in the Ovarian weight (3.14 ± 0.03 versus 2.58 ± 0.06), number of ova (6.00 ± 0.37 versus 11.17 ± 0.40).
Conclusion(s): Short-term administration of a low-dose of aspirin to mice at proestrous causes the following changes:
significant increase in ovarian weight with an increased congestion in the uterus and ovary. A significant decrease in
ovulatory rate with no effect on serum gonadotropins and estradiol levels.
Key wards: Low-dose aspirin, uterine and ovarian morphological changes, and ovulation rate.
Acetylsalicylic acid was first synthesized in effective in the prevention and treatment of
1897. The product was called aspirin. In 1971, cardiovascular disease when given in low-doses
Vane described the mechanism of aspirin action, (2–6). Furthermore, it has been demonstrated that,
showing that it inhibits the enzyme low-dose aspirin increased the weight of newborns
cyclooxygenase, thus preventing prostaglandin in pregnant patients with fetal growth retardation,
(PG) synthesis (1). Because PGs participate in it is also used to prevent idiopathic fetal growth
almost all human body systems, it is not surprising retardation and to improve placental and fetal
that, a drug with a mechanism of action related to blood flow in women with pre-eclampsia (7–
them should have multiple therapeutic uses. Since 9).Non-steroidal Anti-inflammatory Drugs
its beginnings, aspirin has been used as an (NSAIDs) acts largely through inhibition of cyclo-
analgesic, anti-inflammatory, and an antipyretic oxygenase (COX), an enzyme that catalyses the
agent. It is well known that low-dose aspirin is formation of prostaglandin from arachidonic acid
(10). Aspirin, causes irreversible inactivation of
Correspondence: Prof. Adnan AL- Janabi, Nahrain University, cyclo-oxygenase(COX) acting on both COX-1 and
Email alnahrain_ online@yahoo.com COX-2 (11–13). In mice, COX-2 is rapidly
Vol. 12, No. 1, 2007 Al-Janabi et al. Effects of aspirin on ovulation rate 47induced by L.H in cumulus and granulosa cells of aim of determining the minimal effective dose of
the ovarian follicle, with some evidence of an aspirin .The preparation used was Aspegic®
oocyte contribution to COX-2 expression (14). (Laboratories Synthelabo, France). In all cases
Although there are differences between primate (Treated animals=36 mice), the dose of the drug
and rodent ovaries (15), COX-2 inhibition was administered twice daily, starting at the
produces the same results in both species. proestrous phase of the estrous cycle. The 1st drug
Prostaglandins such as PGE2 and PGF2α are injection was administered at 9 a.m. and the 2nd at
critical in the process of ovulation (16), as proved 1 p.m. on the same day (subcutaneously).
by failure of ovulation in deficient phenotype mice Depending on the outcome of the results of this
(COX-2-1-) (17). In rats, the treatment with PG experiment; prevention of ovulation and
synthetase inhibitors is associated with: failure of morphological macroscopic changes in the
follicular rupture; follicular hyperemia and edema reproductive system, the dose level of 7.5mg/kg of
(18); and suppressed synthesis of PGF2α (19). b.w. was adopted as the suitable minimal dose for
Low-dose aspirin reduced follicular level of all other experiments in this study, (minimal
PGF2α and PGE2, and the anti-inflammatory agent inhibitory dose). Animals (36 mice) that received
must be completely abolishes the preovulatory distilled water served as control for the
elevation in PG in mature follicle in order to totally experimental group. Treated mice were
inhibit ovulation in rabbits (20). In women, it has anaesthetized at the metestrous phase (24-84 hours
been reported that, treatment of young women with after the last injection), (using Nembutal®, sodium
aspirin to treat inflammatory joint disease, induces pentoparbital - Alevetra – West Germany) and
luteinized unruptured follicle syndrome (LUF) blood was collected through cardiac puncture and
(21). Moreover, aspirin or NSAIDs are associated sera were prepared for the hormonal assay. After
with the recurrent development of this syndrome in the killing the whole reproductive system was
young women receiving treatment with those drugs quickly removed after killing, and was immersed
to treat ankylosing spondilytis and rheumatoid in a Petri-dish filled with in vitro medium (IVF)
arthritis, and normal ovulation can be resumed in (Universal IVF medium, medicult, Denmark) kept
these patients following drug withdrawal (22). at 37°C. Both ovaries were quickly dissected out,
cleared from surrounding non-ovarian tissue and
weighed using electronic precision balance
MATERIALS AND METHODS (Sartorious – Switzerland). The uteri were then
quickly dissected out slightly at the tubouterine
All experiments were performed on mature junction from one end and immediately close to the
female Swiss white mice, 15-16 weeks old with a internal orifice of the cervix from the other end,
body weight ranging from 25-30g. The mice were they were cleared from surrounding non-uterine
obtained from the colony of the animal house of tissue and were dried from IVF fluid using filter
the institute for embryo research and infertility paper and they were then weighed using electronic
treatment, University of Baghdad. They were kept precision balance (23). Then ova flushed from the
in an air-conditioned room (22-24°C) with an oviduct to determine the ovulation rate.
automatically controlled photoperiod (14 hours The following hormones were assayed using
light and 10 hours darkness). Mice were fed the (Radio-immunoassay kits– RIA –), the assay was
standard balanced pelleted diet presented with tap carried out in the hormonal lab. of Al-Kindy
water "ad libitum". Before experimentation all Teaching Hospital: Luteinizing hormone (LH),
mice were left for at least three weeks for mIu/mL; Follicle stimulating hormone (FSH),
adaptation. Mice then showed at least three mIu/mL. These two mentioned hormones, kits used
consecutive regular cycles were included in the were provided by: (cis biointernational, France).
study (n=72 mice). Mice were then divided into While 17β-estradiol (E2), pg/mL. Kit used was
treated and control groups (n=36 mice each). provided by: (Immunotech, Beckmann coulter
Treated animals were allocated into 6 equal dose company, France), by using LKB-Wallac,
group(25, 20, 15,10,7.5 and 5 mg/Kg b.w) with the Minigamma 1275 Finland. The dissection of the ovaries
48 Al-Janabi et al. Effects of aspirin on ovulation rate MEFSJFigure 1. The genital system of mature female mouse at Figure 3. Ovarian and uterine weight changes associated with
metestrous phase (control group), showing the ovary (O), aspirin administration at proestrous phase.
uterine horn (U) and oviduct (OD).
from the oviduct was carefully performed, flushing RESULTS
of ova was done using tuberculin syringe (30
gauges) with blunt ended needle filled with 1mL of A- Morphological changes:
IVF medium was inserted into the fimbriated end
of the oviduct and held in place with fine surgical Aspirin administered to cycling mice on the
forceps. Then recovered ova were kept in a Petri day of proestrous and killing on the metestrous
dish with IVF medium. And their number was phase (i.e. during the period of ovulation),
counted along with their quality using a dissecting induced several morphological changes in the
microscope under 2.5X, 16X magnification (24). ovaries and uteri as follows: Ovaries appeared
enlarged, cystic, congested with visually
Statistical Analysis prominent blood vessels with few corpora
hemorrhagica. On the other hand, the ovaries of
Collected data were analyzed using SPSS control animals, appeared less congested, filled
version 10.0 for windows (SPSS, Chicago, Illinois, with corpora hemorrhagica and blood vessels on
USA). Differences of means between groups were the surface of the ovaries were less prominent.
examined by student t-test, P. valueTable 2. Hormonal changes associated with Aspirin
administration to mature cycling female mice (7.5mg/Kg.bw)
on day of proestrus phase and killed on day of metestrus
phase.
Hormones Serum Level
Control Experimental
LH mIu/mL 4.65 ± 0.05 4.24 ± 0.09
FSH mIu/mL 8.97 ± 0.11 8.67 ± 0.24
Estradiol pg/mL 21.80 ± 0.18 21.67 ± 0.28
Figure 4. Mature ova flushed from the oviduct of mice treated Values are mean ± standard error (SEM), (n=6 animals/group).
with (7.5mg-Kg.bw) aspirin and examined in the metestrous
phase, showing signs of normality: presence of first polar
body (PB) with round and even cytoplasm (65X). DISCUSSION
C-Number of flushed ova and their quality: Ovarian and uterine morphological changes
obtained in the experimental animals namely:
Concerning effect of aspirin on ovulation rate: intense organ congestion, cystic appearance of the
administration of aspirin to the cycling mice ovaries as compared to the control are clear
caused a highly significant (P < 0.001) decrease in indication of a pronounced inflammatory response.
the number of ova flushed from the oviduct, as This result is an expected outcome of a combined
compared with the control(6.00±0.37 and effect of both normal physiological morphological
11.17±0.40) for experimental and control group changes associated with ovulatory process (25),
respectively. Administration of aspirin caused no and vascular changes induced by aspirin treatment
deterioration in the quality of the flushed ova, e.g.: vasodilatation and increased vascular
since no ova with fragmented polar body, or permeability (26). All these inflammatory changes
degenerative oocyte, or cytoplasmic fragmentation, occurred normally during ovulation are thought to
or vacuolated cytoplasm, or amorphic shape, or be mediated mainly by: prostaglandins (PGE2 and
post mature oocyte were seen (Figure 4). PGI2) and histamine (27, 28). Moreover PGF2α is
a well known factor in terminating inflammatory
D-Hormonal changes: changes associated with ovulation (29), and its
synthesis is stimulated by gonadotropins (30)
No significant changes were seen in the serum which are known to increase during this period
level of both gonadotropins(FSH and LH) as well as (31). Based on these interactions we are inclined to
estrogen(E2) in the aspirin treated mice, as compared suggest that inhibition of PGF2α by aspirin
to the control, when blood was examined on the day administration may have blocked or prevent the
of metestrous phase (Table 2). termination of the inflammatory process and
allowing it to extend longer than normal. The
cystic appearance of ovaries of experimental
Table 1. Changes in the weight of the ovaries and uteri, animals are due to failure of ovulation of certain
associated with aspirin administration to mature cycling female
mice (7.5mg/Kg.bw) on day of proestrus phase and killed on day
number of graafian follicles which has been turned
of metestrus phase. into luteinized unruptured follicles (32, 34).
Inhibition of Prostaglandin synthesis in the
Organ Weight (mg) preovulatory follicles by aspirin hinders
completion of the ovulatory process leading to
Control Treated
Ovary 2.58 ± 0.06 3.14 ± 0.03* failure of follicular rupture a hence formation of
Uterus 27.24 ± 0.25 27.37 ± 0.17* cysts (35). Formation of these cysts has a positive
impact on ovarian weight, since in ovulation, there
Values are mean ± standard error (SEM), (n=6 animals/group). is normally release of not only the ova but also the
*
= significant changes (P < 0.001)
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