Abstract

Background: As the United States Drug Organization reports, more than 38 million people around the world are addicted to amphetamine and its derivatives.
Objectives: The present research investigates the effects of methamphetamine on the biochemical parameters of the amniotic sac in pregnant rats and the ovaries of their offspring.
Methods: 60 adults female Wistar rats were divided into 6 groups: control group, sham group, the group receiving a dose of 1,2,3, and 4 mg/kg of amphetamine, respectively. Amniotic fluid was separated to check the amount of protein, carbohydrates, fats, phospholipids, and urea. Tissue sections were prepared from the ovary with H&E staining.
Results: Testosterone and LH levels in groups M1 and M3, unlike FSH, showed a significant difference with the control group. M1 and M3 groups have the lowest amount of glucose compared to the control group, and the highest amount of protein belongs to M1 group. Histological results showed that the underlying substance and growing follicles in the tissue section of the ovary of group M were destroyed and the theca layer was separated from the follicle and several follicular cells were removed and released into the atrium. The results of gene expression indicated that the overall ratio of HOX and TUBB8 genes is 0.11 and 0.52, respectively.
Conclusions: The injection of methamphetamine has significant effects on the biochemical parameters of the amniotic sac, increases or decreases the activity of the ovary and oogenesis, and disrupts the quality of ovulation and maturation of the oocytes.

Keywords:Methamphetamine; Amniotic Fluid; Ovary; Hematoxylin-Eosin

Abbreviations: ADHD: Attention Deficit Hyperactivity Disorder; HOX genes: Homeobox Genes; TG: Triglyceride; GnRH: Gonadotropin-Releasing Hormone

Background

As the United States Drug Organization reports, more than 38 million people around the world are addicted to amphetamine and its derivatives. Amphetamines are a group of central nervous system stimulants that were discovered more than 100 years ago. Amphetamine and many of its derivatives are very diverse in structure and physiological effects. The most important effect of amphetamines is increasing the extracellular concentration of catecholamines and monoamines (epinephrine, norepinephrine, dopamine, and serotonin) through the integration of secretory vesicles into the presynaptic membrane. Although this drug was freely available in the past, today it has become a controlled drug with limited therapeutic applications in the treatment of attention deficit hyperactivity disorder (ADHD) and narcolepsy [1]. It also reduces sleep, fatigue, and appetite, increases the pain threshold, and improves attention and performance [2].

Methamphetamine is a derivative of amphetamines, which like amphetamine causes the release of neurotransmitters [3]. Methamphetamine affects the pleasure circuit of the brain and changes the amount of certain neurotransmitters in the synapses. Methamphetamine is chemically similar to dopamine and other neurotransmitters such as norepinephrine. Therefore, it naturally causes the release of dopamine and epinephrine in the brain. Methamphetamine enters the nerve terminal by passing through the nerve cell membrane and returns dopamine and norepinephrine from the synapse to the nerve terminal by the transporter molecule. Methamphetamine enters the dopamine and norepinephrine vesicles inside the terminal and causes their release. Therefore, methamphetamine, amphetamine, and cocaine are amine releasers. The enzyme in the cell naturally renews dopamine and norepinephrine, but methamphetamine blocks this pathway. Thus, excess neurotransmitter is released by transporters in the synapse. A high concentration of dopamine synapses causes a feeling of pleasure and euphoria. Excess norepinephrine is responsible for the increased alertness and fatigue-relieving effects of methamphetamine.

Amniotic fluid is an amazing and complex composition inside the amniotic sac. Although this liquid has antimicrobial effects, it contains vital compounds such as nutrients, hormones, and numerous growth factors that support the growth of the human embryo. Its total volume reaches 500-2500 milliliters at the end of pregnancy. It is located in the amnion cavity (a kind of membrane bag) and surrounds the fetus. Low and high amniotic fluid volumes are called oligohydramnios and polyhydramnios, respectively [4]. Some researchers treated 12 women with symptoms of polyhydramnios with indomethacin at the rate of 2.2-3 mg per kilogram, of which 11 responded to the treatment, and their delivery was delayed by 6.4 weeks. Indomethacin had no significant side effects. Another group of researchers treated 8 women with symptomatic polyhydramnios at a gestational age of 28.6 weeks with indomethacin. The average termination of pregnancy in them was 39 weeks [5].

Homeobox genes (as HOX genes) as essential regulators of anterior-posterior (A-P) axial pattern development is a group of highly conserved genes that are. HOX genes comprise a family of regulatory molecules that encode highly conserved transcription factors. Molecular and genetic evidence recently shows that HOX genes are expressed along the anterior-posterior axis and control cell morphogenesis and differentiation during embryonic axial development. HOX genes play a similar role in determining developmental fate in distinct regions of the female reproductive tract, where they regulate the developmental axis during the embryonic period. HOX genes also confer a specific identity to the developing endometrium during the menstrual cycle in adults. HOX gene expression is regulated by sexual steroids, and this regulated expression plays an important role in endometrial growth and endometrial receptivity. HOX genes encode proteins that act as transcription factors. Several factors affect female fertility, including natural aging and several disease processes. However, two processes are necessary for fertility in any normal woman: ovarian follicular maturation and embryo implantation.

HOX genes in vertebrates are involved in the processes of ovarian follicular maturation and embryo implantation. Ovarian follicle development is a complex process with many transcription factors [6-9].

Microtubules are complex dynamic polymers of heterodimers composed of alpha-tubulin polypeptide and beta-tubulin polypeptide such as TUBB8. TUBB8 among the isotypes encoding β-tubulin is unusual because it is only present in mammalian species. TUBB8 is specifically expressed in the oocyte and early embryo, where it accounts for nearly all expressed β-tubulin. This gene encodes a special isotype of beta-tubulin as the main component of the ovum and spindle of the early embryo that is only found in mammals. Mutations in TUBB8 cause the arrest of oocyte maturation. A Genetic defect in TUBB8 gene class VIII is responsible for oocyte maturation, fertilization, and early fetal growth arrest. The TUBB8 gene has 4 coding exons and can be mapped to chromosomes p 3, 10, and 15 based on the alignment of the TUBB8 sequence with the genomic sequence (GRCh38). Heterozygous or de novo mutations in the TUBB8 gene mainly cause metaphase I arrest through dominant-negative effects, although some arrests also occur early in embryonic development. However, heterozygous, homozygous, or compound mutations may result in fertilization failure and arrest of early embryonic development. Oocyte maturation and subsequent mitosis are important periods for normal fetal development. Aneuploidy caused by abnormal spindle accumulation is an important cause of premature fetal arrest.

Mutant TUBB8 with oocytes showed that mitosis is more sensitive to the effect of spindle assembly than meiosis. As recent research has shown, two bipolar spindles are formed in the zygote and then independently assemble the maternal and paternal genomes in mice and humans. This mechanism of spindle assembly provides a potential rationale for the sensitivity of zygote spindles to microtubule gene mutations and illustrates the difference between mitosis and meiosis mechanisms during oocyte and zygote transformation. This TUBB8 mutation may provide a unique perspective on the differences between meiotic and mitotic spindle assembly in the mother-to-zygote transition in mammals. These findings showed the essential role of TUBB8 in human oocyte maturation and female fertility and increased our understanding of TUBB8 mutations’ interference with zygote development [10,11]. Since the use of narcotic drugs including methamphetamine can have a potential effect on amniotic fluid, the expression of effective genes in the mother and fetus, tissues, hormones, and other factors, this research investigates the effects of methamphetamine on the biochemical parameters of the amniotic sac in pregnant rats and the ovaries of their offspring.

Methods

Laboratory Animals

This experimental study purchased 60 adults female Wistar rats, weighing 180-220 grams, from the research center of Royan Research Institute and transferred them to the laboratory of the research center of North Tehran Azad University. The animals had enough access to water and food during the study and were kept under standard light cycle conditions of 12 hours of light and 12 hours of darkness with a temperature of 23±2 degrees Celsius. It is noteworthy that all the ethical principles of working with laboratory animals were observed based on the ethical guidelines of the international association.

Preparing Methamphetamine Solution

Methamphetamine was gained from the anti-narcotics headquarters. Then it was dissolved in distilled water in the required concentrations and injected intraperitoneally to the animals at specified hours.

This research was carried out in two stages:

At first, each female rat was placed in a separate cage next to a male rat to mate the animals. The observation of vaginal plaque was supposedly on the first day of pregnancy. Pregnant mothers were grouped in 6 injection groups for one week before pregnancy until delivery as follows:
G1: female pregnant rats that received normal nutrition.
G2: female pregnant rats that received distilled water.
G3: female pregnant rats that received methamphetamine at a dose of 1 mg/kg in two doses in the morning and in the evening.
G4: female pregnant rats that received methamphetamine at a dose of 2 mg/kg in two doses in the morning and evening.
G5: female pregnant rats that received methamphetamine at a dose of 3 mg/kg in two doses in the morning and evening.
G6: Female pregnant rats that received methamphetamine at a dose of 4 mg/kg in two doses in the morning and evening.

Then the neonates were grouped into 6 groups of 10 as follows:
G1A: offspring of rats that received a normal diet.
G2B: offspring of female pregnant rats that received distilled water.
G3C: offspring of female pregnant rats that received methamphetamine at a dose of 1 mg/kg in two doses in the morning and evening.
G4D: offspring of female pregnant rats that received methamphetamine at a dose of 2 mg/kg in two doses in the morning and evening.
G5E: offspring of female pregnant rats that received methamphetamine at a dose of 3 mg/kg in two doses in the morning and evening.
G6F: offspring of female pregnant rats that received methamphetamine at a dose of 4 mg/kg in two doses in the morning and evening.

Measuring Biochemical Parameters

All the animals were anesthetized using ketamine and xylazine after the end of the experiment period, and direct anesthesia was performed by a syringe from their hearts. The blood samples were centrifuged at 3500 rpm for 15 minutes, and their serum was separated and stored at -20 degrees Celsius until the tests. The level of testosterone hormones was measured with a kit (manufactured by IBL, Germany), LH, FSH (manufactured by Biomerio, France), liver enzymes AST, ALT, and glucose, RBC, and WBC tests. The amnion sac was separated immediately after the mother rat was killed, the weight of each of them was calculated and the contents inside the sac were stored and transferred to the laboratory to examine the amount of protein, carbohydrates, fats, phospholipid, and urea.

Histological Investigations

Ovarian tissue samples were separated from the rats under study to examine the tissue changes, placed in Buen’s for 24 hours, and then fixed in a 10% formalin solution. The tissue samples were stained with Hematoxylin-eosin after the processing steps and then the tissue sections were examined under a microscope.

Real-Time PCR

This study extracted total RNA with the help of Triazole according to the manufacturer’s instructions. The amount of light absorption at wavelengths of A260/ 280 with the help of a bio photometer and zinc loading 2.5% agarose gel to evaluate the quality control of the extracted RNA. The cDNA synthesis was performed by Themo Fisher kit according to the manufacturer’s instructions. The Real-Time PCR reaction was carried out in three steps using Biofact 2X Master Mix and SYBR Green method according to the manufacturer’s instructions. Table 1 gives the primers used in the experiment.

Statistical Analysis

The collected data were analyzed using SPSS software, the Kruskal-Wallis Test, and 1-sample k-s. Then the relevant histograms were drawn using Excel software and p 0.05 was a significant difference.

Results

The following tables and graphs give the data for measuring the biochemical parameters of the groups under study. Table 2 examines the biochemical parameters in the group receiving methamphetamine with a dose of 1 mg/kg (M1). The amount of SGOT was significantly higher than the control group in the groups receiving methamphetamine, and SGPT did not show a significant difference (Figure 1). The values of FSH, LH, and testosterone did not show a significant difference in the control group. PH was neutralizing in groups receiving methamphetamine. The triglyceride level in the groups tested with methamphetamine was significantly lower than the control group. LH values in the group receiving methamphetamine with a dose of mg/kg2 (M2) decreased compared to the control group, but this decrease was not significant. There was no significant difference between the FSH and testosterone values in the tested groups and the control group. The TG and LDL values in the group receiving 2 mg of methamphetamine were significantly lower than the control group (Figure 1). Injection of methamphetamine with a dose of 3 mg/kg (Table 1) had no significant effect on FSH, LH, testosterone, and other biochemical parameters (Table 2).

A significant decrease in TG level was also seen in this group, as with other doses of methamphetamine. LH decreased in the group receiving 4 mg of methamphetamine compared to the control group, but this decrease was not significant (Table 1). LDL in the tested groups significantly decreased compared to the control group (Figure 1). Changes of LH hormone in the M3 group are equal to 0.604 and more than other groups receiving methamphetamine. Meanwhile, the M2 group has the lowest amount of LH hormone. As the average data of testosterone hormone measurement show, the average changes of testosterone hormone in the M3 group is equal to 4.914 and more than other methamphetamine-receiving groups. Table 3 presents the amount of glucose in amniotic fluid samples. As it is clear, the glucose level did not show a significant difference between the tested groups and the control group. M1-1, M1-3, and M3-3 groups have the lowest amount of glucose compared to the control groups (Figure 2).On the contrary, as Table 3 reveals, the amount of protein showed a significant difference between the methamphetamine and control groups. The protein level in the tested groups increased significantly compared to the control group (Figure 2). The highest amount of protein was for the M1-1 group.

Histological Results of the Ovary

The following were the results of the present study, which was performed on multiple slides of ovarian tissue in each group:
The tissue section of the ovary in the control group shows healthy follicles in different stages of development. Growing follicles with a regular layer of granulosa cells are observable in the control group. The corpus luteum can be seen in large quantities in these samples, which shows the ovary in the ovulation stage or the beginning of Metestrus (Figure 3). The underlying substance and growing follicles have been destroyed in the tissue section of the ovary of group M. Unlike the control group, this group shows growing follicles with an irregular and confused layer of granulosa cells. The theca layer is separated from the follicle and several follicular cells are plucked and released into the atrium (Figures 3 & 4).

Expression Results of HOX and TUBB8 Genes

The quality of the RNA samples was checked by agarose gel electrophoresis to make sure that the extracted RNA was not degraded. Two bands 8S and 18S were visible in the examination of the gel, which reveals the lack of RNA degradation. Conventional PCR was performed to confirm the primers after cDNA synthesis. This study normalizes HOX and TUBB8, which are the target genes, by reference gene GAPDH. The relative expression of the target genes, on 8 rats, was compared between experimental groups, including S, M1, M2, M3, M4, and M5 groups, through the CTΔ method and CTΔ 2 calculation. First, the CTs of HOX and TUBB8 genes in the experimental groups were compared with the reference gene according to the data of Real-Time PCR. The overall ratio of HOX and TUBB8 genes compared to GAPDH is 0.11 and 0.52, respectively (Table 4). The expression of HOX and TUBB8 genes was investigated in five experimental groups, including S, M1, M2, M3, M4, and M5, with 4 different doses of methamphetamine. As the results show, as the dose of methamphetamine increases, the expression of these genes decreases (Figure 5).

Discussion

Methamphetamine has become recently an attractive drug in several countries because of its ease of manufacture and low cost compared to other profitable drugs. Methamphetamine is an illegal psychostimulant drug of the amphetamines. Methamphetamine is a highly addictive drug that is slowly absorbed over a long period. It is usually used by young people and teenagers who are of reproductive age. Previously, many studies had proven the negative effects of morphine and cocaine, but the use of synthetic drugs, including methamphetamine, has increased today in developed and developing countries. The exact mechanism by which amphetamine or methamphetamine leads to erectile dysfunction in men is not completely known. Experimental studies on rodents show the mechanisms of methamphetamine’s effect on men’s fertility potential, which include altered hormonal profiles, oxidative stress, DNA damage to sperms, and abnormal spermatogenesis. Methamphetamine also negatively affects the seminiferous epithelium, including germ cell degeneration and apoptosis. Methamphetamine, as one of the chemical narcotics and mimics neurotransmitters, can easily occupy the relevant receptors and cause disorders in the reproductive system through continuous use. As the present study showed, intraperitoneal injection of methamphetamine caused significant changes in biochemical parameters. Another study by Pontes et al. [12] has shown that MDMA increases liver enzymes in the blood [2]. Thus, Moon et al. concluded that MDMA caused tissue changes by inhibiting ATP synthase and increasing the level of liver transaminases [13]. Beitia et al., by investigating the adverse effect of intraperitoneal injection of MDMA, concluded that this substance increased the activity of all liver enzymes, especially ALT, and increased cholesterol levels. Glycogen content showed in their study a significant decrease, which was associated with a decrease in serum glucose level [3].

The data obtained from the examination of liver enzymes AST and ALT in M groups receiving methamphetamine show in the present study a significant increase compared to the control group. The amount of glucose in the groups receiving methamphetamine showed a significant decrease compared to the control group, which is consistent with the results of the above studies. Lipids may play roles in central nervous system function associated with drug addiction. Serum lipids influence relapse to heroin use. Previous research has investigated changes in serum lipid levels in heroin and opium users. The group addicted to heroin and opium showed a significant decrease in serum triglyceride (TG) levels. Another study found that rats showed a significant increase in serum TG after injection of methamphetamine. Little research has assessed serum lipid levels in large sample sizes of methamphetamine-dependent individuals; the relationship between serum lipids and methamphetamine craving is unclear. Another study observed that the levels of TG, TC, and glucose (GLU) were significantly decreased in methamphetaminedependent patients. Methamphetamine-dependent patients suffer from cognitive deficits and abnormal metabolic activity that affects nutritional status. This situation is worsened by a severe decrease in oral health in methamphetamine-dependent patients, leading to improper chewing and poor digestion. This explains why TG, TC, and BMI levels were reduced in methamphetaminedependent subjects. The triglyceride level in the groups receiving methamphetamine, in all doses, decreased compared to the control group, which was consistent with the above studies. Similarly, the glucose level in the methamphetamine-treated groups was lower than in the control group, and this result is consistent with the results of other studies.

The physiology of gonadotropin hormone regulation is under the control of gonadotropin-releasing hormone (GnRH). Therefore, their effect on gonads and sex hormones is very complex and occurs through hormones, neurotransmitters, receptors, and multiple mechanisms in the body. Compounds with a structure similar to these substances such as amphetamine, ecstasy, morphine, and other drugs can have intensifying or weakening effects on the regulation system of sex hormones, gametogenesis, and fertility [14]. Injecting morphine into pregnant mice by affecting the pituitary-gonadal axis in a study by Vathy et al. caused a decrease in LH and FSH and secondary follicles, and a decrease in fertility [15]. The slow growth and development of secondary follicles as an effect of morphine is because morphine and other opioids, by affecting the hypothalamus, cause a decrease in the GnRH hormone, followed by a decrease in the transfer of these hormones to the pituitary gland through the capillary shunt. Continuous injection of morphine at intervals of 6 hours, during pregnancy, with an increasing concentration of 2.5-10 mg/kg per day, causes 34% abortion and weight loss in newborn rabbits [16]. As the results of another study showed, the administration of 0.66 mg/kg morphine to pregnant sheep did not cause significant changes in umbilical blood flow or fetal oxygen consumption but decreased the amount of umbilical vein glucose. Indeed, the administration of morphine to the mother changes the fetal glucose balance because of the reduction of placental penetration [4].

Opioid drugs do not act similarly. MDMA can stimulate follicular activity [17]. Knigge et al. showed in their studies that ecstasy has a great affinity with histamine receptors and, since histamine increased the LH response to GnRH secretion, it led to the premature rupture of the follicles and the earlier release of the ovum [18]. Ecstasy also leads to increased release of serotonin from nerve terminals. Likewise, the use of serotonin-releasing drugs leads to an increase in the release of prolactin in them. An increase in prolactin leads to a decrease in FSH gonadotropes [5]. As the results of Meller et al. showed, the long-term use of another similar substance called Ritalin (methylphenidate C14H19NO2) at the beginning of sexual puberty causes concern because of its effect on the function and structure of the female reproductive system (ovaries). The greatest effect of this drug is in the release of GnRH from the hypothalamus, and it leads to premature maturation of the gonads by stimulating gonadotropes, which usually leads to premature and abnormal activity of the gonads, disruption of gametogenesis, and the production of abnormal gametes [19].

Methamphetamine caused many changes in the ovarian tissue, which include the destruction of the underlying substance and growing follicles, confusion and reduction of the granulosa cells around them, weakening and thinning of the layer of theca in the follicle graft, and the removal of the underlying substance of the ovary from the normal state. These results are in line with those of the study of Safi et al.

The physiology of gonadotropin hormone regulation is very complex and occurs through multiple mechanisms, neurotransmitters, hormones, and receptors. Because of the structural similarity of narcotic compounds such as morphine, ecstasy, and amphetamine with these agents, they can easily replace them and exert weakening or intensifying effects on gametogenesis, sex hormones, and the reproductive system in general [20, 21]. As some studies show, secondary follicles and the fertility rate decreased following the injection of morphine into pregnant mice and its effect on the pituitary-gonadal axis, LH, and FSH. Continuous injection of morphine at 6-hour intervals, during pregnancy, with an increasing concentration of 2.5-10 mg/kg per day, causes 34% abortion and weight loss in rabbits [22, 23].

Research showed that ecstasy has a great affinity with histamine receptors, and since histamine increases the LH response to GnRH secretion, it leads to the premature rupture of follicles and the premature release of egg cells [24]. The results of another study showed that the administration of 0.66 mg/kg of morphine to pregnant sheep did not cause significant changes in umbilical blood flow or fetal oxygen consumption, but the amount of glucose in the umbilical vein decreased. Indeed, administration of morphine to the mother changes fetal glucose balance because of decreased placental penetration [25]. The effect of methamphetamine on causing morphological disorders in egg cells and reducing fertility was a proven fact [12]. Other researches show that the extract of plants such as poppies on the reproductive process in both male and female genders causes fundamental and irreparable disorders in babies after birth, so babies whose parents are under the influence of related drugs get pulmonary and hormonal disorders that cause the loss of lung tissue cells in babies after birth. The children of these parents suffer from hormonal disorders after puberty and their ability to reproduce in the next generations decreases greatly [26, 27].

Another study has proven the effect of morphine on rat ovulation blocking it, and irregular zing the menstrual cycle in humans [6]. The destructive effects of methamphetamine on the ovarian tissue were probably because of one of its most dangerous effects, which is the increase in body temperature that affects the structure of the ovarian tissue. Likewise, methamphetamine can cause changes in the blood vessels of the ovary by releasing norepinephrine from the sympathetic nerve endings, and eventually cause damage to the follicles [7,9].

Conclusions

As the data of the present study showed, injection of methamphetamine caused significant changes in the amount of LH hormone, testosterone hormone, liver enzyme ALT (SGPT), glucose level, and protein level. Therefore, the injection of methamphetamine, even in low doses, by several mechanisms such as binding to receptors, increases or decreases the activity of the ovary and oogenesis and disrupts the quality of ovulation and maturation of oocytes.

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