the effect of l-carnitine and metformin on histomorphology...

BIHAREAN BIOLOGIST 14 (1): 16-24 ©Biharean Biologist, Oradea, Romania, 2020

Article No.: e191305 http://biozoojournals.ro/bihbiol/index.html

The effect of L-carnitine and metformin on histomorphology characteristics

of uterus in mice with polycystic ovary

Sara AHMADI SHOKOOH and Mehri AZADBAKHT*

Department of Biology, Faculty of Sciences, Razi University, Kermanshah, Iran.

* Corresponding author, M. Azadbakht, E-mail: [email protected]

Received: 29. January 2019 / Accepted: 03. June 2019 / Available online: 19. May 2019 / Printed: June 2020

Abstract. The polycystic ovary (PCO) is related to low levels of serum L-carnitine, which has a tri-methylamine molecule that

essential roles in energy production, oxidative stress and glucose metabolism. Polar metformin has a positive charge, low molecular

weight and multiple function in a number of tissues including muscle, liver, pancreas, adipose tissue, hypothalamus, pituitary and

genital glands. Intracellular and metabolic disorders in PCO may have complex effects on the uterus. The aim of this study was to

investigate the effect of L-carnitine and Metformin on histomorphology characteristics in mice following induction of PCO. PCO

was induced by daily injections of testosterone enanthate (1 mg per 100 g, s.c., for 4 weeks). NMRI mice (16-18 days old) were

divided into eight groups (n=6 per group): Intact (I), I+L-carnitine, I+metformin, I+L-carnitine+metformin, PCO, PCO+L-carnitine,

PCO+metformin, PCO+L-carnitine+metformin groups. Mice were treated with 250 mg/kg, i.p., L-carnitine daily for 4 weeks and

250 mg/kg, i.p., Metformin daily for 2 weeks. At the end of the treatment, the mice were sacrificed by cardiac puncture. Blood

samples were collected, centrifuged and plasma samples hormone analysis with ELISA kits. After dissecting, the uterus was isolated

for histomorphologic examination. Data were analysed using one-way analysis of variance (ANOVA) and Tukey’s test, differences

considered significant at (p 0.05). The body weight and concentration of testosterone decrease and endometric thickness, muscle

thickness, characteristics of glands and total uterine diameter increase significantly, in the PCO+L-carnitine, PCO+Metformin and

PCO+L-carnitine+metformin versus PCO group. L-Carnitine and Metformin both improve the characteristics of the uterus

suggested as a therapeutic supplement in the treatment of PCO.

Key words: L-carnitine, metformin, polycystic ovary, uterus, mice.

Introduction

The uterus has three layers, which together form the uterine

wall. These layers from inside to outside are endometrium,

myometrium and perimetrium. The endometrium consists of

stromal and epithelial parts (luminal and glandular) with

separate functions that contribute to the regulation of uterine

homeostasis (Cunha et al. 1989). Ovarian sexual steroids,

17beta-estradiol and progesterone, play essential roles in

regulating uterine proliferation, stromal-epithelial commu-

nication and differentiation in the preparation for pregnancy

(Simitsidellis et al. 2016). The first in 1949 was the associa-

tion between endometrial cancer and polycystic ovary syn-

drome (Navaratnarajah et al. 2008). Intracellular and meta-

bolic disorders in this syndrome may have complex effects

on the uterine endometrium (Giudice 2006). In women with

the polycystic ovary, the endometrium is exposed to pro-

longed stimulatory effects of estrogen without progesterone

due to chronic ovulation, which results in expression of en-

dometrium androgen receptors (Elliott et al.2001, Iatrakis et

al. 2006). In addition, in the endometrium, insulin-like

growth factor-1 (IGF-1) is produced (Park et al. 2011). The

presence of prolonged estrogen without progesterone, in-

creased insulin and androgens and high levels of free (IGF-

1), further increase the activity of endometrial mitosis, which

ultimately leads to increased hyperplasia (increased number

of cells) and possibly endometrial cancer (Park et al. 2011,

Franks 1995, Cheung 2001). The increase in the concentra-

tion of the sex hormones binding hormones and endometrial

aromatase regulation, androgen enhancement and increased

insulin increases the potential for further changes in Ne-

oblastic endometrium (Giudice 2006, Balen 2001, Speroff &

Fritz 2005). Increasing androgen induces morphological

changes in the uterus that cause of miscarriage (Sir-

Petermann et al. 2002, Tuckerman et al. 2000). This increase

leads to the development of pre-cancerous structures with

the induction of apoptosis in the uterine endometrium, lead-

ing to abortion (Sir-Petermann et al. 2002, Elia et al. 2008).

The most important treatment in PCOS is the use of drug

protocols, hormones, or the use of assisted reproductive

techniques (ART) such as In Vitro Fertilization (IVF) and In

Vitro Maturation (IVM) (Cobin et al, 2005). The use of ovula-

tion inducers attempt to revitalize the menstrual cycle and

improve fertility weight loss exercise and ovulation inducers

such as clomiphene citrate, gonadotropins, insulin-

susceptible drugs, ovarian laparoscopy are essential for

PCOS (Igwegbe 2013). Metformin with a chemical formula

(C5H4N11) is a class of biologically active substance that is a

commonly used antidiabetic agent and a commonly used

Drug for the treatment of type 2 diabetes (Salomaki et al.

2013). Metformin inhibits liver gluconeogenesis and reduces

insulin levels in the bloodstream. The use of this drug in pa-

tients with PCOS improves insulin's effect, reduces the level

of androgens, and leads to regular menstrual flow (Ve-

lazquez et al. 1994, Bertoldo et al. 2014). The primary effect

of metformin is the reduction of liver glucose production by

activating the AMP-enabled protein kinase enzyme (AMPK)

(Katzung et al. 2004). Metformin, although reducing intesti-

nal glucose, increases insulin sensitivity in peripheral tissues

(Yildiz et al. 2008). The use of metformin in patients with

polycystic ovary syndrome decreases the premature and

pre-antral follicles. Metformin leads to improved follicular

development and increases the percentage of yellow corpus-

cles and decreases cystic structure, which leads to an in-

crease in ovulation and fertility rates (Di Pietro et al. 2015).

Metformin is also used to produce androgen low ovaries, fa-

cilitate pregnancy and reduce abortion in the first trimester

in patients with PCOS (Jakubowicz et al. 2002). Metformin

The effect of L-carnitine and metformin on uterus in mice with PCO

17

improves all surrogate markers of endometrial receptivity in

PCOS patients, without difference between patients who had

the favourable or unfavourable reproductive outcome (Pal-

omba et al. 2005). L-carnitine plays a major role in energy

production, oxidative stress, glucose metabolism and me-

tabolism of fatty acids by facilitating the transfer of long-

chain free fatty acids to the mitochondrial matrix and mak-

ing them available for beta-oxidation, which is the most effi-

cient route for energy production (Borum 2012, Fenkci et al.

2008). Many studies have also reported a significant decrease

in markers of oxidative stress, including MDA and inflam-

matory factors, such as IL-6 and TNF-a, following L-

carnitine intake (Duranay et al. 2006, Li et al. 2012, Bavari et

al. 2016). L-carnitine administration increased oocyte volume

in PCOS mice, which could be due to the fact that L-carnitine

has an important role in glucose metabolism and its intake

can reduce insulin resistance in obese PCOS patient (Samimi

et al. 2016). Studies demonstrating the antiapoptotic action

of L-carnitine (Bavari et al. 2016, Kanter et al. 2010). As a free

radical scavenger, L-carnitine prevents follicular atresia by

reducing oxidative stress and inhibiting mitochondrial dys-

function (Kanter et al. 2010). Incubating the oocytes and em-

bryo of patients with endometriosis in the intraperitoneal

fluid has shown increase the apoptosis in the embryo's.

While conjugation with L-carnitine can significantly reduce

the apoptosis of the embryos (Mansour et al. 2009). Also, L-

carnitine and L-acetyl-carnitine can improve the mobility

and quality of chromatin in the sperm (Aliabadi et al. 2012).

Material and Methods

Animal: Forty-eight immature female average 17 NMR mice with

days of age were obtained from the Pasteur Institute of Tehran and

were kept in the Razi University animal house under standard con-

ditions (12-h dark–light cycle, mean (± s.d.) temperature 21±2ºC)

with an adequate supply of food and water. They were fed with a

standard laboratory mouse diet (Dane-e-pars Co., Kermanshah,

Iran). The animals were allocated in groups (eight) of six individuals

per cage. This experiment was approved by the Laboratory Animal

Care Committee of Razi University. The experimental procedures

were carried out in accordance with international guidelines for care

and use of laboratory animals.

Experimental design: In the first phase of this study, the mice

were divided into two groups; intact mice (I, 24 mice) and mice with

the polycystic ovary (PCO, 24 mice). In the second phase of the

study, each initial group was further divided into four treatment

groups; the intact group (I, 6 mice), intact receiving L-carnitine

group (I+LC,6 mice), intact receiving Metformin group (I,6 mice), in-

tact receiving L-carnitine and metformin group (I+LC+Met, 6 mice),

polycystic ovary group (PCO, 6 mice), polycystic ovary receiving L-

carnitine group (PCO+ LC, 6 mice), polycystic ovary receiving Met-

formin group (PCO+Met, 6 mice), polycystic ovary receiving L-

carnitine and Metformin group (PCO+LC+Met, 6 mice). In which

mice were injected daily with 250mg/kg, i.p., L-carnitine (Sigma-

Aldrich) for 4 weeks (Duranay et al. 2006, Ricci et al. 2002, Skrtic et

al. 2011) and 250mg/kg, i.p., Metformin for 2 weeks.

Induction of the polycystic ovary (PCO): Mice (16-18 days old)

were randomly divided into two groups (n=24 per group): In the

PCOS group, PCO was induced by daily subcutaneous injections of

testosterone enanthate (Androne; Caspian Tamin) dissolved in ses-

ame oil at a dose of 1 mg per 100 g body weight for 4 weeks (Skrtic et

al. 2011, Kalhori & Azadbakht 2015). Mice in the non-PCOS group

were injected with an equivalent volume (1 mL) of sesame oil for 4

weeks.

Preparation of the vaginal smear: Throughout the treatment pe-

riod, vaginal smears were daily assessed in order to determine the

stages of the estrous cycle. The amount of 50 mL saline was used for

vaginal aspiration. The smeares were stained with Papanicolaou

stain (Hubscher et al. 2005). Vaginal smears from each mouse were

examined under a light microscope (magnification_10) and the stage

of the estrous cycle classified as proestrus, estrus, metestrus and die-

strous. Biochemical analysis: At the end of the treatment, blood sam-

ples were obtained and centrifuged at 3000g for 5 min at room tem-

perature; the serum was collected for determination of testosterone

concentration. The serum concentration of testosterone was meas-

ured using commercially available kits according to the manufactur-

ers’ instructions: the testosterone ELISA kit (Catalogue no. DKO002;

Dia Metra, sensitivity 0.07 ng/mL, intra- and interassay CVs 5.4 and

15% respectively).

Preparation of tissue slide: Mice were sacrificed at days (70-72)

by a cardiac puncher. After the uterus isolated were fixed in Bouin’s

fixative for 15 h, after which they were dehydrated in an ascending

series of ethanol (50– 100%), cleared in xylene by an automatic tissue

processor (Histokinette; Leica) and finally embedded in paraffin. To

prepare isotropic uniform random (IUR) sections, the ‘isector’ meth-

od was used. Briefly, cylindrical paraffin blocks were rotated in a

random manner and serially cut into (5m) sections. The sections

were stained using haematoxylin and eosin (Merck) and 12 sections

per animal were randomly selected for histological examination

(Karbalay-Doust & Noorafshan 2012, Mahmoodi et al. 2014). Using

the Dino-capture software, uterine changes were examined.

Statistical analyses: The statistical analysis was performed by

one-way ANOVA and Tukey’s test. All values were expressed as

means ± standard error (s.e.). Test values with a p 0.05 were consid-

ered significantly different. All data analyses were performed using

Minitab statistical software (version 17.1, 2013).

Results

Estrous cycle: A regular estrous cycle of (4-5) days was seen

in the intact group, while results of vaginal smear cytology

showed that after PCO induction, all mice stopped in the di-

estrous phase with smears containing mostly leucocyte cells

(Fig. 1d). Treatment in the PCO+L-carnitine, PCO+ Metfor-

min, PCO+L-carnitine +metfoemin could restore cytology of

vaginal smear to normal and similar to the intact group. The

effectiveness of the beginning of the third week was

achieved. For this reason (Table 1) represents the Estrous cy-

cle from days 43 to 56 of the experiment.

Body weight: In the PCO group increased the body

weight compared to the intact group. In the PCO+ L-

carnitine+ Metformin group body weight loss was greater

than that of PCO+ L-carnitine group and the PCO+ Metfor-

min group (Table 2).

Quantitative studies characteristics of the uterus: The in-

duction of PCO decreased endometrial thickness, surface ep-

ithelial cell core diameter and surface epithelial cell count, as

well as increase surface epithelial cell length (p≤0.05) in

comparison with the intact group (Table 3). In the PCO+L-

carnitine group increase endometrial thickness, surface epi-

thelial cell core diameter and surface epithelial cell count but

decreased surface epithelial cell length (p 0.05) comparison

with the PCO group (Table 3). In the PCO+metformin group

increased endometrial thickness, surface epithelial cell

length, surface epithelial cell core diameter and surface epi-

thelial cell count (p 0.05) comparison with the PCO group

(Table 3). In the PCO+L-carnitine+metformin group

S. Ahmadi Shokooh & M. Azadbakht

18

Table 1. The results of the study estrous cycle in the groups. Days (43-46).

PCO I Treatment

days PCO+LC+Met PCO+Met PCO+LC PCO I+LC+Met I+Met I+LC I

Metestrous Metestrous Diestrous Diestrous Diestrous Metestrous Diestrous Metestrous 43

Metestrous Proestrous Diestrous Diestrous Proestrous Metestrous Metestrous Metestrous 44

Proestrous Estrous Diestrous Diestrous Estrous Proestrous Metestrous Metestrous 45

Estrous Metestrous Diestrous Diestrous Metestrous Metestrous Metestrous Proestrous 46

Metestrous Metestrous Proestrous Diestrous Metestrous Metestrous Proestrous Estrous 47

Metestrous Diestrous Estrous Diestrous Diestrous Metestrous Estrous Metestrous 48

Diestrous Estrous Metestrous Diestrous Proestrous Estrous Metestrous Metestrous 49

Proestrous Metestrous Metestrous Diestrous Estrous Metestrous Metestrous Diestrous 50

Estrous Metestrous Diestrous Diestrous Metestrous Metestrous Diestrous Proestrous 51

Metestrous Metestrous Proestrous Diestrous Metestrous Diestrous Proestrous Estrous 52

Metestrous Proestrous Estrous Diestrous Metestrous Proestrous Estrous Metestrous 53

Metestrous Estrous Metestrous Diestrous Proestrous Estrous Metestrous Metestrous 54

Proestrous Metestrous Metestrous Diestrous Estrous Metestrous Metestrous Metestrous 55

Estrous Metestrous Metestrous Diestrous Metestrous Metestrous Metestrous Proestrous 56

Table 2. Comparison of the average weight of animals

between different groups.

GROUPS character

treatment

Average weight of mice(gr)

I I 27.000.62abc

I+LC 25.631.14abc

I+Met 24.1250.71bc

I+LC+Met 23.870.58c

PCO PCO 28.370.26a

PCO+LC 27.750.70a

PCO+Met 27.1250.81ab

PCO+LC+Met 26.370.49abc

*a/b: Significant vs within groups (p≤0.05), Data were expressed as

(mean ± SEM)

increased endometrial thickness, surface epithelial cell

length, surface epithelial cell core diameter and surface epi-

thelial cell count (p 0.05) comparison with the PCO group

(Table 3). In the PCO group, reduced the myometrium

thickness, circular and longitudinal muscle thickness

(p≤0.05) in comparison with the intact group (Table 4, Fig 2).

In the PCO+L-carnitine group showed an increase in myom-

etrium thickness, longitudinal and circular muscle thickness

(p≤0.05) compared with the PCO group (Table 4, Fig 2). In

PCO+metformin group increased in myometrium thickness,

longitudinal and circular muscle thickness (p≤0.05) com-

pared with the PCO group (Table 4, Fig 2). In PCO+L-

carnitine+metformin group increased in myometrium thick-

ness, longitudinal and circular muscle thickness (p≤0.05)

compared with the PCO group (Table 4, Fig 2). Comparison

of large gland between the intact and PCO groups showed

that the induction of PCO, destruction reduced the gland ep-

ithelial cell length, count and gland total diameter in the

PCO group compared to the intact group (p≤0.05). The gland

epithelial cell core diameter and gland lumen diameter did

not change in the PCO group compared to the intact group

(Table 5). Comparison of large glands showed that PCO+L-

carnitine group increased the gland epithelial cell length,

Figure 1. Mice Estrous cycle. A) Proestrous

phase, B) Estrous phase, C) Metestrous

phase, D) Diestrous phase. The implications

indicated are: E: nucleated epithelial cell, C:

horny epithelial cell, L: leukocyte.


19

Table 3. Comparison of the mean endometrium characteristics of the uterus.

GROUPS characters

treatment

ET(m) SEL(m) SECD(m) SEC(on 50 m)

I I 306.693.49cd 15.960.70d 6.480.46a 14.500.34bcd

I+LC 294.231.78cd 23.870.97a 6.510.34a 12.660.33cd

I+Met 395.1027.60ab 20.240.66b 6.240.30ab 16.660.88b

I+LC+Met 203.552.74ef 16.350.33cd 6.110.21ab 15.660.33bc

PCO PCO 172/0016.41f 17.830.56bcd 5.080.27b 11.330.49d

PCO+LC 256/6013.71de 15.351.11d 5.710.08ab 12.830.30cd

PCO+Met 332.721.47bc 19.790.23bc 5.740.13ab 15.000.73bc

PCO+LC+Met 432.204.50a 23.851.11a 6.110.14ab 17.001.21b

*a/b: Significant vs within groups (p≤0.05), Data were expressed as (mean ± SEM)

*ET: endometrial thickness, SEL: surface epithelial cell length, SECD: surface epithelial cell core diameter, SEC: surface epithelial cell count

Table 4. Comparison of the mean myometrium characteristics of the uterus.

GROUPS characters

Treatment

MT(m) CMT(m) LMT(m)

I I 210.115.55d 102.301.67cd 107.811.25c

I+LC 145.146.25e 80.377.04d 64.763.83d

I+Met 294.411.18b 146.783.33ab 147.613.06ab

I+LC+Met 209.250.80d 43.114.12bc 90.8010.49cd

PCO PCO 174.243.62de 82.702.35d 91.543.48cd

PCO+LC 258.4011.47bc 151.0019.19ab 107.348.59c

PCO+Met 256.494.84c 135.023.91bc 121.317.48bc

PCO+LC+Met 357.059.79a 180.723.77a 176.339.61a

*MT: myometrium thickness, CMT: circular muscle thickness, LMT: longitudinal muscle thickness


diameter and gland total diameter compared to the PCO

group (p≤0.05). The gland epithelial cell core diameter and

lumen diameter of the gland showed no significant change

(Table 5). Comparison of large glands showed that in

PCO+metformin group increased count and gland total di-

ameter and gland epithelial cell core diameter compared to

the PCO group (p≤0.05); The gland epithelial cell length and

gland Lumen diameter showed no significant change com-

pared to the PCO group (Table 5). Comparison of large

glands showed that in PCO+L-carnitine+metformin group

increased the gland epithelial cell length, count and gland

total diameter and decreased gland epithelial cell core diam-

eter compared to the PCO group (p≤0.05). The gland lumen

diameter showed no significant change (Table 5). Compari-

son of small glands showed that induction of the polycystic

ovary decreased gland epithelial cell length and gland total

diameter of the small glands and increased lumen diameter

(p≤0.05) in comparison with the intact group (Table 6).

Comparison of small glands showed that in PCO+L-

carnitine group increased gland epithelial cell length and

decreased gland lumen diameter (p≤0.05) compared with the

PCO group. Gland epithelial cell core, count and gland total

diameter showed no significant change (Table 6). Compari-

son of small glands showed that in PCO+met-formin group

decreased gland total diameter and gland lumen diameter

(P≤0.05) compared with the PCO group. Gland epithelial cell

length and gland epithelial cell core diameter and a count

showed no significant change (Table 6). Comparison of small

glands showed that in PCO+L-carnitine+metformin group

increased gland epithelial cell length and gland total diame-

ter, decreased gland lumen diameter (p≤0.05) compared

with the PCO group. Gland epithelial cell core diameter and

count showed no significant change (Table 6). The induction

of PCO decreases the uterine total diameter and gland count

(p≤0.05) to comparison with the intact group (Table 7). In the

PCO+L-carnitine group increase uterine total diameter and

gland count compared to the (p≤0.05) PCO group (Table 7).

In the PCO+metformin group increase uterine total diameter

and gland count compared to the (p≤0.05) PCO group (Table

7). In the PCO+L-carnitine+metformin group increase uter-

ine total diameter and gland count compared to the (p≤0.05)

PCO group (Table 7).

Qualitative studies characteristics of the uterus: Histo-

logical examination of the intact group (I) showed that stro-

ma was low in density and intercellular space was high. Eo-

sinophilic cytoplasm and multi-nucleated cells and dead

cells were scattered. The blood vessels were low. Small di-

ameter glands were visible. Surface epithelium and cylindri-

cal glands were simple. The nucleus of the epithelial cells of

the testicle and stretched, there are two types of bright and

dark cells in the epithelium (Fig. 2a, 3a and 4a). Histological

sections of the PCO group showed increased folds and de-

creased space lumen. Cells with frothy appearance (subject

to degraded changes) were observed. In the epithelial, the

sticky Mucosa covered the surface of the epithelial cells. The

number of secreted glands was very low and in decline. Se-

cretion of the glands was reduced. The muscles were deplet-

ed. Significant destruction occurred in epithelial cells. Very

low mitotic activity and vascularization increase in endome-

trium and myometrium (Fig. 2e, 3e and 4e). The results of

the histological examination showed, I+L-carnitine group,

stroma was low in density and intercellular distance was


20

Table 5. Comparison of the mean large glands characteristics of the uterus.

GROUPS characters

Treatment GECL(m) GECCD(m) GECC(m) GLD(m) GTD(m)

I I 14.960.38ab 5.08±0.05ab 31.17±1.22bcd 20.69±1.55a 52.15±2.40bc

I+LC 12.47±0.61abc 5.22±0.09ab 26.00±1.36d 36.21±8.74a 44.72±5.59c

I+Met 11.870.69bc 5.690.12ab 29.503.25cd 36.007.5a 51.541.53bc

I+LC+Met 14.88±0.44ab 5.60±0.22ab 25.66±0.33d 24.32±0.51a 53.00±0.36bc

PCO PCO 10.26±0.25c 4.98±0.13ab 29.83±.05cd 25.49±0.17a 46.29±0.45c

PCO+LC 15.04±0.39ab 5.22±0.02ab 39.33±3.18ab 29.40±1.03a 70.70±0.16a

PCO+Met 9.440.63c 5.840.30a 36.671.08abc 27.040.32a 61.690.59ab

PCO+LC+Met 16.03±1.99a 4.84±0.13b 44.00±1.18a 29.34±0.83a 66.69±0.70a


*GECL: gland epithelial cell length, GECCD: gland epithelial cell core diameter, GECC: gland epithelial cell Count, GLD: gland lumen

diameter, GTD; gland total diameter

high. L-carnitine increased a lot of death and inset. Increase

in blood vessels. The muscles have been reduced and spaced

apart. Adhesive mucosa was seen at the epithelial cell sur-

face. The gland is seen with a large lumen. Cell death had

reduced the density of epithelial cells (Fig. 2b, 3b and 4b). In

the PCO+L-carnitine group, it also showed that L-carnitine

increased cell death in the stroma and suppressed inflamma-

tory and the epithelial cells are arranged and their density is

reduced to that of the PCO group. The glands still had a

foamy appearance, but the epithelium had somewhat lost its

foamy appearance (Fig. 2f, 3f and 4f). The results of the his-

tological examination showed, I+metformin group, the

stroma is dense and intercellular space is low and the cells

were very close together. The surface epithelium was pseu-

dostratified. The nucleus of the surface epithelium cells was

pulled but the nucleus of the epithelium cells gland was

nodular in the basal area. Surface epithelium cells shorter

than the epithelium cells gland. In this group, the number of

Figure 2. Transects of the uterus of the

mice, number and size of the glands,

thickness of the myometrium; A) I

group, B) I+LC group, C) I+Met

group, D) I+LC+Met group, E) PCO

group, F)PCO+LC, G) PCO+Met,

H) PCO+ LC+Met (H&E staining).

Signs on Figures; G: gland, M: my-

ometrium, L: Lumen.


21

Table 6. Comparison of the mean small glands characteristics of the uterus.

GROUPS characters

Treatment GECL(m) GECCD(m) GECC(m) GLD(m) GTD(m)

I I 15.22±0.62a 4.57±0.82a 17.67±2.47a 8.51±1.07ab 31.29±2.02a

I+LC 15.81±0.41a 5.40±0.14a 13.00±0.44a 5.29±0.42d 30.42±1.83ab

I+Met 13.670.61ab 5.870.13a 14.160.30a 7.530.52abc 30.751.22ab

I+LC+Met 15.80±0.87a 14.42±8.57a 15.30±0.22a 8.18±0.10abc 31.20±0.26a

PCO PCO 9.06±.05c 11.45±6.59a 16.50±2.13a 9.13±0.12a 27.22±0.41ab

PCO+LC 11.30±0.45bc 5.12±0.19a 15.00±0.36a 6.44±0.06bcd 29.70±0.25ab

PCO+Met 9.650.55c 4.760.10a 12.660.21a 6.200.05cd 36.000.08b

PCO+LC+Met 13.44±0.18ab 5.95±0.25a 13.00±0.25a 8.23±0.20abc 31.44±0.06a

*GECL: gland epithelial cell length, GECCD: gland epithelial cell core diameter, GECC: gland epithelial cell count, GLD: gland lumen

diameter, GTD; gland total diameter


glands was greater but not very active. The diameter uterus

lumen was reduced but the uterus total diameter increased

(Fig. 2c, 3c and 4c). While in PCO+metformin group, met-

formin injection causes the glands to warp and twist, Demi-

dov’s reaction, increased blood vessels and inflammatory re-

action. Low cell stroma and dead cells were visible (Fig. 2g,

3g and 4g). The results of the histological examination

showed, I+L-carnitine+metformin group, there was a death

induced by L-carnitine, but there was no effect on secretion.

The thickness of the endometrium was lower. The length of

the epithelial cells was shorter than the control group and

the number of epithelial cells was increased. Epithelial cells

had a much longer gland than the luminal epithelial and had

a larger lumen. The number of glands has been reduced in

total. The myometrium was also depleted (Fig. 2d, 3d and

4d). While in PCO+L-carnitine+metformin it caused the cells

to have no foaming appearance, but death was seen in them.

The number of glands was higher than the polycystic group.

Figure 3. Transects of the uterus of

the mice, blood vessels, cell density;

A) I group, B) I+LC group, C)

I+Met group, D) I+LC+Met group,

E) PCO group, F)PCO+LC, G)

PCO+Met, H) PCO+ LC+Met (H&E

staining). Signs on Figures; G:

gland, M: myometrium, L: Lumen.


22

Table 7. Comparison of the mean characteristics of the uterus.

GROUPS characters

Treatment UDT(m) GN(on 440 m)

I I 1242.66±5.30b 8.16±0.44e

I+LC 1018.00±35.52cd 17.16±0.03c

I+Met 1416.7051.32a 30.671.20b

I+LC+Met 945.93±7.78d 7.00±0.44ef

PCO PCO 610.80±21.96e 4.83±0.54f

PCO+LC 1034.50±46.42cd 8.33±0.21e

PCO+Met 1120.4047.28bc 13.660.21d

PCO+LC+Met 1500.75±5.36a 34.50±0.61a

*UTD: uterine total diameter, GN: gland count

*a/b: Significant vs within groups (p≤0.05), Data were expressed as (mean ±

SEM)

The muscles were similar to the control group. Surface epi-

thelium and cylindrical glands were simple (Fig. 2h, 3h and

4h).

Biochemical analysis: In the PCO group increased serum

testosterone levels compared with the intact group (p≤0.05).

In the PCO+L-carnitine group did not significantly change

the serum testosterone levels comparison PCO group. In the

Table 8. Comparison of average testosterone concentration be-

tween different groups.

GROUPS characters

Treatment TC(ng/ml(

I I 5.66±0.19c

I+LC 6.43±0.21c

I+Met 7.130.92c

I+LC+Met 8.44±2.38c

PCO PCO 15.01±1.75a

PCO+LC 11.00±0.02a

PCO+Met 10.550.65b

PCO+LC+Met 8.24±2.57c

*TC: testosterone concentration

*a/b: Significant vs within groups (p≤0.05), Data were expressed as

(mean ± SEM)

PCO+metformin group decrease serum testosterone levels

comparison PCO group (p≤0.05). In the PCO+L-

carnitine+metformin group decreased (p≤0.05) the serum

testosterone levels comparison PCO group and similar to I

group (Table 8).

Figure 4. Cutaneous transection of

the uterus of the mice, epithelial

cell length (E) and mitotic forms

(M) and apoptosis. A) I group, B)

I+LC group, C) I+Met group, D)

I+LC+Met group, E) PCO group,

F)PCO+LC, G) PCO+Met, H)

PCO+ LC+Met (H&E staining).

Signs on Figures; G: gland, M: my-

ometrium, L: Lumen.


23

Discussion

Women with the polycystic ovary often show metabolic ab-

normalities like obesity, hyperinsulinemia, insulin re-

sistance, angiogenesis, dyslipidemia, hyperandrogenism and

the risk of cardiovascular disease and type 2 diabetes (Oak-

ley et al. 2011). In the present study, PCO was induced in

mice by daily injections of testosterone enanthate for 4

weeks. These mice had reproductive and endocrine charac-

teristics of PCO, such as obesity, hyperandrogenism and

disorder estrous cycle which have also been reported in pre-

vious studies (Skrtic et al. 2011, Kalhori & Azadbakht 2015).

The findings showed that the estrous cycle in mice consists

of four stages: Proestrous, Estrous, Metestrous and Die-

strous. The studies carried out to confirm the results (Ca-

ligioni 2009, Marcondes et al. 2002). Davoudi and colleagues

showed that the effect of estradiol on uterine tissue in mice

increased folding and decreased uterine lumen space and

degeneration and significant necrosis in epithelial cells, as

well as reduction of endometrial thickness and myometrium

thickness and decreased mitotic activity (Davoudi et al.

2015). Studies have been done on women with the polycystic

ovary showed that endometrial thickness of the uterus de-

creased in these patients (Ricci et al. 2002). In the PCO group

decreased the thickness of the endometrium, decreased the

thickness of the uterus, decreased a number of glands and

epithelial cells and ultimately decreased the total uterus di-

ameter. It can be concluded that the polycystic ovary, having

an effect on the thickness of the endometrium and the num-

ber of uterine glands that are important factors in embryo

acceptance, reduces the uterine acceptability of the uterus for

implantation and reduces fertility. Studies by Di Pietro, et al.

(2015) showed that metformin can restore and regularize the

menstrual cycle, modify follicle growth, ovulation induction

and fertility in patients with insulin-resistant PCOS. Studies

by Sander, et al. (2011) showed that metformin modulates

the ovarian steroidogenesis during the luteal phase, and in-

duces ovulation in women with PCOS, also improves the

vascularity of these patients it turns out. In a study by Tan et

al. (2004) that investigated the effect of metformin on reduc-

ing PCOS symptoms, regardless of insulin resistance, met-

formin reduced levels of high blood sugar and also reduced

metastasis and hyperplasia of the uterine endometrium he

does. Stadtmauer et al. (2001) showed that women with the

polycystic ovary have no symptoms of endometrial thick-

ness and uterine envelope in the metformin-treated group.

Zhang et al. (2016) experiments on rats with the polycystic

ovary showed that their treatment with metformin reduced

the growth factor of connective tissue growth in the uterus

and ovary Fenkci and colleagues in a study on women with

polycystic ovaries showed that total L-carnitine levels were

low in these patients (Fenkci et al. 2008). In the

PCO+Metformin increased the thickness of the endometri-

um and uterus and increased the number and size of the

uterus and ultimately increased uterine diameter toward the

PCO group. Fenkci and colleagues in a study on women

with polycystic ovaries showed that total L-carnitine levels

were low in these patients (Fenkci et al. 2008). Experiments

on male rats showed that L-carnitine and L-acetyl-carnitine

increase sperm motility and the quality of spermatic carti-

lage (Ricci et al. 2002). The mechanism of the effect of L-

carnitine has an effect on the structure of microtubules and

to prevent their depolymerization. Microtubules are part of a

cellular skeleton made up of tubulin units. Microtubules are

generally associated with the attached proteins or MAPs,

which phosphorylation of these proteins by cyclin kinases

results in degradation of the microtubule structure. L-

carnitine neutralizes the anticoagulant effect of TNF-α and

reduces the level of damage to DNA. L-carnitine increases

the number of blastocyst cells, which may help to expand

blastocyst and thinness the thickness of the Zona pellucida

and successfully exit for implantation (Khanmohammadi et

al. 2016). The amount of embryo apoptosis decreases with

the incubation of the embryo of the mouse with L-carnitine.

L-carnitine protects cells against apoptosis by inhibiting ac-

tinomycin-D activity, which is an apoptotic inducer

(Mansour et al. 2009). In the PCO+ L-carnitine, the group in-

creased the thickness of the endometrium and uterus and in-

creased the number and size of the uterus and ultimately in-

creased uterine diameter toward the PCO group. It can be

concluded that L-carnitine also can improve the polycystic

ovary by improving endometrial and glands status in com-

parison with the PCO group. The polycystic ovary can alter

the uterus and hormonal levels. The interaction of L-

carnitine and metformin is in contrast with the effect of pol-

ycystic ovaries and to improve its complications. The simul-

taneous injection of these two drugs has a much greater ef-

fect than the injection of each of these drugs. Simultaneous

administration of L-carnitine and metformin would restore

normal ovarian-uterine cycles and greatly reduce the com-

plications of the polycystic ovary on the reproductive sys-

tem. Also better results in increasing endometrial thickness

and uterine muscle gains in the group receiving L-carnitine

and Metformin than those in the polycystic group, which

leads to the development of the estrous phase, which can in-

dicate ovulation stimulation.

Acknowledgement. The authors thank Dr. Manouchehr souri for

technical guidance during this research.

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