Nicotine and endometrial decidual growth in pseudopregnant rats

Abstract

Purpose

The present study aims to investigate the effects of nicotine on the endometrial decidual growth and levels of estrogen and progesterone in pseudopregnant rats.

Methods

Pseudopregnancy (pc) was induced in cyclic Sprague‐Dawley rats by sterile mating. Subcutaneous injection of nicotine tartrate (7.5 mg/kg/day) was scheduled from day 1 through day 5, day 5 through day 9 or day 1 through day 9 of pc. In another group of pseudopregnant rats, concomitant treatment of nicotine tartrate concurrently with progesterone (2 mg/day) was scheduled from day 1 through day 9 pc. Control groups received subcutaneous injections of vehicle only. Endometrial decidualization was induced on day 5 pc. On day 10 pc, animals were sacrificed.The degree of decidual growth and circulating levels of estrogen and progesterone were measured.

Results

The decidual growth in all the first three nicotine‐treated groups of animals was significantly reduced, particularly in the animals treated with nicotine from day 1 through day 9 pc. Plasma estrogen levels were significantly elevated in animals treated with nicotine from day 1 through day 9 pc. Conversely, levels of plasma progesterone were found to be significantly attenuated in the same group of nicotine‐treated animals compared to controls. Exogenous replacement of progesterone, however, caused a higher degree of endometrial decidualization compared to the nicotine‐treated group but it was slightly less than when compared to control.

Conclusions

In conclusion, nicotine‐induced progesterone deficiency with a corresponding elevation of estrogen may possibly attenuate the degree of endometrial decidualization in pseudopregnant rats.

Introduction

The prevalence of tobacco smoking has shown a worrying trend especially in females of reproductive age group despite the adverse effects associated with it [1]. Nicotine, as one of the primary components of tobacco smoke, is known to be harmful to health [2]. Epidemiological, clinical and experimental studies have shown that tobacco smoke containing nicotine has deleterious impact on various stages of female reproduction starting from folliculogenesis up to menopause [3]. Nicotine has been shown to retard growth of the follicles via apoptosis [4], disrupts ovarian steroidogenesis [5, 6], impairs embryo development [7], and delays implantation of the fertilized ovum [8]. Tobacco smoking has also been associated with earlier onset of menopause [9].

The normal reproductive functions in females are regulated by complex interactions between the hypothalamus, pituitary and ovaries, which is the hypothalamic–pituitary–ovarian (HPO) axis. The hormones of this axis are important and any slight disruption to these hormones will result in altered reproductive rhythm. The effect of nicotine on female reproduction is usually dose‐dependent and depends on the duration of its exposure [5]. Nicotine has shown conflicting results on the levels of circulating ovarian steroids; namely estrogen and progesterone [5, 6, 10]. Apart from the ovarian steroids, nicotine also affects hypothalamic [11] and pituitary hormones responsible for normal reproductive functions namely luteinizing hormone [11, 12, 13, 14] and prolactin [14, 15, 16, 17, 18] even though the findings were contradictory.

Endometrial decidual growth has been used as a model to study the factors essentially involved in this process [19]. In this process, endometrial stromal cells differentiate in response to blastocyst or artificial stimuli. Decidualized endometrium also represents a maternal component of the placenta [20]. Endometrial decidualization depends on the interplay of the ovarian steroids and some local factors that are produced upon contact with the blastocyst or artificial stimuli [20]. In ovariectomized rats, maximal endometrial decidual growth is achieved with a combination of a low dose of estrogen and progesterone on an estrogen‐primed endometrium [21, 22]. Conversely, a high level of estrogen consistently results in attenuated decidual growth [23]. Maintenance of deciduoma in rats requires progesterone, thus a functional ovarian luteal tissue is essential [20].

Our study was, therefore, designed to investigate the effects of nicotine on the degree of endometrial decidual growth in relation to corresponding changes in the profile of estrogen and progesterone at different durations of treatment.

Materials and methods

Animals

Female Sprague‐Dawley rats weighing 180–220 g were used in this study. Animals had free access to food and drinking water. The room temperature was kept at 25 °C and the light:darkness cycle was maintained at 12:12 h. Strictly 4‐day cyclic rats were used in this study.

Experimental design

Animals were selected randomly. Control and experimental groups had 6 animals in each group. Pro‐oestrus rats were housed overnight with sexually matured vasectomized males at a ratio of 1:1 to induce pseudopregnancy (pc). The following day of oestrus was considered as day 1 of pc. Continuous di‐oestrus vaginal smears confirmed pc. Treatment was scheduled from day 1 through day 5 pc, day 1 through day 9 pc or day 5 through day 9 pc. Nicotine tartrate (Sigma Chemicals) solution was prepared weekly to achieve the concentration of 7.5 mg/kg and injected subcutaneously (sc) twice a day (0900 and 1600 hours). Progesterone 2 mg/day was injected (sc) to another group of pseudopregnant rats concurrently with nicotine from day 1 through day 9 pc. Control animals received subcutaneous (sc) injection of normal saline/oil/both. On day 5 pc, under anesthesia, endometrial decidualization was induced by injecting 0.1 mL corn oil into one of the uterine horns at the utero‐tubal junction exposed via a dorsolateral incision [24]. On day 10 pc, blood samples were collected (under anaesthesia) for the estimation of estrogen and progesterone. Animals were then sacrificed; ovaries, uterine horns and adrenal glands were dissected out, freed from their adhering tissue, weighed and expressed in mg/100 g body weight. Decidualized uterine horns of the animals were processed for histological study.

Hormone assay

Blood samples were centrifuged at 3000 rpm for 15 min, plasma was stored at −80 °C until estimation of estrogen and progesterone by using an automated electrochemiluminescence immunoassay technique (ECLIA) (Roche Elecsys).

Statistical analysis

A Kolmogorov–Smirnov test was done to assess the normality of the data distribution. Normally distributed data were analyzed using Student's t test for comparison between treated and control groups whereas one‐way ANOVA was used to compare data between more than two groups. Data were expressed as mean ± SEM. Skewed data were analyzed using the Mann–Whitney and the Kruskal–Wallis test for comparison between two groups and more than two groups, respectively. Data were expressed as median (interquartile range). The p values of <0.05 were considered statistically significant. The statistical package SPSS for Windows (version 19.0, SPSS Inc., Chicago, IL, USA) was used for all analyses.

Results

Control and nicotine‐treated groups

Weight of the decidualized uterine horn

Compared to controls, the weight of the decidualized uterine horn in all the treated groups of animals was found to be significantly decreased which was particularly pronounced in the group of animals that had nicotine from day 1 through day 9 pc (Fig. 1). Grossly, the endometrial decidualization in the control animals was found to be uniform throughout the entire length of the uterine horn, whereas in the nicotine‐treated groups, the degree of decidualization was reduced and interrupted (Fig. 2).

Figure 1.

Weight of the decidualized uterine horn (mg/100 g body weight). Data expressed as mean ± SEM, **p < 0.01, *p < 0.05

Figure 2.

Gross morphology of the decidualized uterine horns in control (left) and day 1–day 9 nicotine‐treated (right) animals

Histology of the decidualized uterine horn

The histological changes of the decidualized uterine horns of the control and nicotine‐treated animals are shown in Fig. 3. Differentiation of stromal cells into decidual cells in the nicotine‐treated animal was remarkably impaired compared to control animals.

Figure 3.

Photographs of the decidualized uterine horns in control (left) and day 1–day 9 nicotine‐treated (right) animals. The arrow showed the degree of endometrial decidualization from the lumen to the myometrium layer of the uterus. A huge endometrial decidual cell differentiation and growth are shown in the control as compared to the nicotine‐treated animal. H&E staining, ×10

Weight of the ovary

Table 1 shows the weight of the ovary, which was not significantly different from the controls.

Table 1.

Weight of the ovary (mg/100 g body weight)

Groups	Treatment from day 1 through day 5 pc, mean ± SEM	Treatment from day 5 through day 9 pc, mean ± SEM	Treatment from day 1 through day 9 pc, median (IQR)
Control	26.68 ± 0.64	26.75 ± 0.77	29.65 (6.63)
Nicotine	26.14 ± 1.04	26.05 ± 1.14	26.88 (3.16)

Weight of the adrenal gland

Figure 4 shows adrenal gland weight of all the three groups of animals. Compared to controls, the mean weight of the adrenal gland was significantly higher in animals that had nicotine from day 1 through day 9 pc.

Figure 4.

Weight of the adrenal gland (mg/100 g body weight). Data expressed as mean ± SEM, **p < 0.01

Estrogen

Plasma concentration of estrogen showed tendency to be higher following nicotine treatment. It was found to be significant in the group of animals that received nicotine from day 1 through day 9 pc (Table 2).

Table 2.

Plasma levels of estrogen (pmol/L) in the control and the nicotine‐treated group, data expressed as median (IQR), * p < 0.05

Groups	Treatment from day 1 through day 5 pc, median (IQR)	Treatment from day 5 through day 9 pc, median (IQR)	Treatment from day 1 through day 9 pc, median (IQR)
Control	52.62 (12.18)	52.11 (9.13)	52.52 (8.52)
Nicotine	55.93 (15.35)	59.80 (14.32)	56.44 (15.96)*

Progesterone

Compared to controls, concentrations of plasma progesterone did not differ in the groups of animals which received nicotine from day 1 through day 5 pc or which had nicotine from day 5 to day 9 pc. However, the progesterone level was significantly reduced in the group of animals, which was treated with nicotine from day 1 through day 9 pc (Table 3).

Table 3.

Plasma levels of progesterone (nmol/L) in the control and the nicotine‐treated group, data expressed as median (IQR), *** p < 0.001

Groups	Treatment from day 1 through day 5 pc, median (IQR)	Treatment from day 5 through day 9 pc, median (IQR)	Treatment from day 1 through day 9 pc, median (IQR)
Control	332.75 (103.82)	329.05 (92.02)	335.30 (109.90)
Nicotine	310.65 (125.18)	343.30 (140.00)	257.70 (113.55)***

Control, nicotine‐treated and following supplementation with progesterone (2 mg/day) from day 1 through day 9 pc

Comparison of the weight of the decidualized uterine horns, ovaries and adrenal glands among control, nicotine‐treated and nicotine concurrently supplemented with progesterone from day 1 through day 9 pc are summarized in Table 4.

Table 4.

Weight of the stimulated uterine horns, ovaries and adrenal glands (mg/100 g body weight) in control, nicotine‐treated and nicotine‐treated group supplemented with progesterone, data expressed as mean ± SEM

Organ weight (mg/100 g body weight)	Control	Nicotine 7.5 mg/kg	Nicotine 7.5 mg/kg and progesterone 2 mg/daily
Decidualized uterine horn	878.10 ± 37.66	450.18 ± 37.09**	641.95 ± 69.63** ,^
Ovary	27.13 ± 0.75	27.77 ± 1.29	22.06 ± 1.19*,^
Adrenal gland	24.31 ± 2.22	33.30 ± 1.55**	29.10 ± 1.58

** p < 0.01 compared to control, * p < 0.05 compared to control, ^{^} p < 0.05 comparison between nicotine‐treated and nicotine‐treated supplemented with progesterone

Weight of the decidualized uterine horn

Progesterone supplementation to the nicotine‐treated group caused a significant increase in the weight of the decidualized uterine horn when compared to the nicotine‐treated group. However, the weight of the decidualized uterine horn in the progesterone supplemented group was found to be lower than that of the controls (Table 4).

Weight of the ovary

There was no difference between the weights of ovaries in the nicotine‐treated groups as compared with that of the control (Table 4). However, there was a significant reduction in the ovarian weight in the nicotine concurrently supplemented with the progesterone group compared to controls and nicotine‐treated groups, respectively, at treatment duration from day 1 through day 9 pc.

Weight of the adrenal gland

Progesterone supplementation to the nicotine‐treated group caused a slight increase in the adrenal weights. But the weight of the adrenal glands in this group was not significantly different from either the control or nicotine‐treated group.

Comparison of estrogen and progesterone levels between control, nicotine‐treated and nicotine concurrently supplemented with progesterone from day 1 through day 9 pc are summarized in Table 5.

Table 5.

Plasma levels of estrogen (pmol/L) and progesterone (nmol/L) in control, nicotine‐treated and nicotine‐treated group supplemented with progesterone, data expressed as median (IQR)

Level in plasma	Control	Nicotine 7.5 mg/kg	Nicotine 7.5 mg/kg and progesterone 2 mg/daily
Estrogen (pmol/L)	52.58 ± 2.08	60.21 ± 2.95	48.6 ± 5.46
Progesterone (nmol/L)	359.20 ± 12.47	274.66 ± 15.77*	449.75 ± 56.89* ,^

*p < 0.05 compared to control, ^{^} p < 0.01 comparison between nicotine‐treated and nicotine‐treated supplemented with progesterone

Estrogen

There was a decrease in the estrogen level when progesterone was supplemented to nicotine‐treated group compared to nicotine‐treated and control groups, but the decrease was not significant.

Progesterone

There was a significant increase in the progesterone level in the progesterone supplemented nicotine‐treated animals compared to both control and nicotine‐treated groups.

Discussion

Nicotine tartrate injection in this study showed a significant reduction in the weight of decidualized uterine horn as compared to the control when nicotine was given at three different treatment schedules. However, it was most significant when nicotine was scheduled for a longer duration, that is, from day 1 through day 9 pc. Earlier studies also showed that a prolonged treatment duration had a more significant impact [5]. Treatment commenced from day 1 through day 5 pc affected the reproductive processes prior to fertilization whereas treatment commenced from day 5 through day 9 pc affected the post‐fertilization period or the early trimester of pregnancy. The different treatment schedules will affect the production of ovarian steroids at different parts of the pregnancy. Card and Mitchell [23] reported that there was reduction in weight of the decidualized uterine horn in rats when nicotine (5 mg/kg) was administered from day 0 to day 9 pc or from day 0 to day 4 pc; whereas similar treatment from day 5 to day 9 pc failed to show any significant effect on the weight of the decidualized uterine horn compared to the control. However, the possible mechanism of this differential endometrial response to nicotine remains unexplained.

Development and maintenance of the uterine endometrial decidualization are mainly controlled by the circulating ovarian steroids; namely estrogen and progesterone, which in turn are regulated by the hypothalamic and anterior pituitary hormones [20]. Estrogen in the proestrous stage is important in priming the endometrium, followed by progesterone and estrogen released from the corpus luteum [20]. Since progesterone is important in maintaining proliferation, hypertrophy and differentiation of uterine stromal cells [25], an appropriate level of progesterone is essential to initiate and maintain endometrial decidual growth. Studies also showed that a wide range of progesterone levels in the maternal plasma of a pregnant rat would not affect placental growth, embryonic survival and fetal growth [26, 27, 28]. In ovariectomized rats, treatment with various doses of progesterone only produced submaximal decidual growth, but when estrone was added with the progesterone, the degree of decidualization was almost the same as the control [29]. Estrogen is known to potentiate the action of progesterone on decidual growth and survival of the fetus [25, 28]. Estrogen in excess, however, causes fetal mortality, retards growth of the fetus and placenta [26, 30] by inducing progesterone deficiency [31]. The inhibitory action of a high dose of estrogen may also be explained by its property in inducing transforming growth factor mRNA [32]. This in turn encodes a protein that promotes apoptosis [33]. It is, therefore, reasonable to assume that a slight decrease in progesterone level may not cause significant reduction in the degree of endometrial decidualization. However, a combination of alow level of progesterone with a high level of estrogen may retard appropriate endometrial decidual growth.

In our study, plasma progesterone levels in all the three treatment groups tended to be lower than their respective controls, but became significantly lower in the group that was treated with nicotine from day 1 through day 9 pc. Conversely, the estrogen level was significantly higher in the group treated with nicotine from day 1 through day 9 pc. This finding indicates that the low progesterone level together with the high level of estrogen could possibly result in the attenuation of endometrial decidualization.

Regarding the nicotine‐related female reproductive endocrine profile, some studies suggested that cigarette smoke or nicotine inhibits the steroidogenic pathway lowering an endogenous estrogen level when compared to nonsmokers [10, 34, 35, 36, 37]. However, others observed that the level of estrogen remained significantly higher when exposed to cigarette smoke or nicotine [5, 6]. Progesterone levels have been reported to be reduced in the presence of nicotine [5, 6, 38].

In the present study, progesterone supplementation in the nicotine‐treated animals increased the endometrial decidualization as compared to the nicotine‐treated group, but this increase did not reach the degree of decidualization attained by the control group. The plasma progesterone level in this group was significantly higher than the control and nicotine‐treated group whereas the plasma estrogen level was not significant. These changes in the ovarian steroid profiles may be attributed to the increase in decidualization of endometrium. However, the exogenous administration of progesterone with the corresponding changes in the plasma ovarian steroids was not enough in reversing the deleterious effect of nicotine on the degree of endometrial decidualization. This finding indicates that other factors apart from ovarian steroids also play a role in endometrial decidualization.

Prolactin/LH/both is considered to be a luteotropic hormone. It has been reported that prolactin was responsible for the activation of corpus luteum, which will secrete progesterone as a consequence whereas the maintenance of corpus luteal function was the role of luteinizing hormone [39]. Nicotine showed conflicting results on the secretion of prolactin and LH. A low dose of nicotine failed to affect the plasma level of prolactin, whereas a high dose of nicotine resulted in hyperprolactinaemia [14]. Apart from that, there was also a report of low prolactin levels with exposure to nicotine [18]. Studies also showed higher prolactin levels following nicotine treatment [16, 17] and attenuation of prolactin release with chronic nicotine injections [15]. Delayed surge of luteinizing hormone following nicotine injections [12, 13, 40] subsequently resulted in a decreased LH level [41]. It was also reported that the LH level increased after exposure to high dose nicotine [14]. This altered secretion of the anterior pituitary luteotropic hormones in response to nicotine might also cause attenuation of endometrial decidual growth.

As a rapidly differentiating and proliferating cell, the decidua needs competent vascular blood flow to supply the growing cells with all the important nutrients, and, thus, the maintenance of uteroplacental blood flow is important. Uteroplacental blood flow has also been shown to be compromised with high dose nicotine treatment [42, 43, 44, 45]. Such a reduction in blood flow to the rapidly dividing cell involved in endometrial decidualization may lead to attenuation of decidual growth.

Insignificant changes in ovarian weight between the nicotine‐treated and control groups failed to suggest morphological luteolysis. However, exogenous progesterone caused significant ovarian weight loss compared to control and nicotine‐treated groups. The exogenous progesterone could have suppressed the corpus luteum in producing endogenous progesterone via a negative feedback mechanism as suggested in ewes [46] but not in rats [47].

However, a significant increase in adrenal weight in animals with nicotine from day 1 through day 9 pc suggests an activation of the pituitary–adrenal axis. Since nicotine increases levels of plasma corticotropin (ACTH) and corticosterone in a dose‐dependent manner [48, 49, 50], activation of any of the components of the hypothalamic–pituitary–adrenal axis such as, CRH, ACTH, β‐endorphin or glucocorticoids evidently exerts profound inhibitory effects on the pituitary–ovarian axis, which consequently results in defective endometrial decidualization [51]. Intact functioning of the pituitary–ovarian axis is essentially important to maintain the first half of pregnancy in rats [52]. Increases in the level of stress steroids have been implicated in impaired reproductive processes [53, 54] that includes endometrial decidualization.

In conclusion, nicotine‐induced attenuated decidual growth is possibly mediated through multiple mechanisms. There is no doubt that the hormones from the hypothalamus, anterior pituitary and ovaries do have a major role in this attenuation; however, the nicotine‐induced stress‐related deactivation of the HPO axis should equally be emphasized as a significant cause of reproductive dysfunction.

Acknowledgments

This research is supported bya Fundamental Research Grant Scheme, Ministry of Higher Education, Malaysia (Grant code: RMI/ST/FRGS 5/3/Fst (18/08). The ethical approval was granted by the Ethics Committee, University Teknologi MARA, Malaysia.

Conflict of interest

All the authors declare that they have no conflict of interest.