Hypothyroidism Affects Uterine Function via the Modulation of Prostaglandin Signaling

Simple Summary A article proved that, in rats with PTU-induced hypothyroidism, the E2 level as well as the expression of the uterine-receptivity factors homeobox A10 and osteopontin was decreased. Additionally, we observed changes in the expression of PGE2, PGF2α, and PGI2 signaling pathway elements, and changes in the concentrations of those prostaglandins in uterine tissue. The results suggest that hypothyroidism may interfere with the prostaglandin signaling pathway, which may further result in a reduction in uterine receptivity. Abstract Thyroid hormones control the functions of almost all body systems. Reproductive dysfunctions, such as abnormal sexual development, infertility, or irregularities in the reproductive cycle, might be associated with thyroid disorders. Uterine receptivity is the period when the uterus is receptive to the implantation of an embryo. During the receptivity period (implantation window), a newly formed blastocyst is incorporated into the uterine epithelium. Prostaglandins are well-known primary mediators of pathological conditions such as inflammation and cancer but are also essential for the physiology of female reproduction. The aim of this study was to evaluate the possible relationship between hypothyroidism and changes in the prostaglandin signaling pathways in the uterus and in the process of uterine receptivity in a rat model. The results show that hypothyroidism impaired uterine receptivity by decreasing the level of E2 as well as decreasing the expression of the uterine-receptivity factors homeobox A10 and osteopontin. Moreover, hypothyroidism caused changes in the expression of elements of the prostaglandin E2, F2α, and I2 signaling pathways and changed the levels of those prostaglandins in the uterine tissue. The results suggest that the mechanisms by which hypothyroidism affects female reproductive abnormalities might involve the prostaglandin signaling pathway, resulting in a subsequent reduction in uterine receptivity.


Introduction
Thyroid hormones control the functions of almost all the body's systems. They stimulate growth and development, affect metabolism, and are essential for the proper function of the central nervous system, cardiovascular system, and immune system, as well as influencing the reproductive system [1][2][3][4][5]. Thyroid hormones regulate the secretion of the main reproductive hormones-estradiol (E2) and progesterone (P4). These hormones are necessary for the maturation and development of the oocytes, prepare the endometrium The role of prostaglandins in embryo implantation is indisputably essential [48,49]. Poor endometrial receptivity during embryo implantation has been linked to reduced prostaglandin synthesis in the human endometrium [50]. On the other hand, it is also known that hypothyroidism can cause problems with fertilization and implantation, miscarriages, and late-pregnancy complications [10,11]. The aim of this study was to evaluate the possible relationship between PTU-induced hypothyroidism and changes in prostaglandin signaling pathways in the uterus and in the process of uterine receptivity in a rat model.

Animals
All the experimental procedures were approved by the Local Animal Care and Use Committee in Olsztyn, Poland (Agreement No. 40/2015/DTN).
Female Wistar rats aged 8-10 weeks were kept in the Animal Laboratory of the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences in Olsztyn. The rats were conventionally housed. They were divided into two groups: the control group (n = 20) and the experimental group with induced hypothyroidism (n = 20). The control group was fed ad libitum. Over the 90 days, the study group, besides the normal diet, also received 0.05% 6-propyl-2-thiouracil solution (PTU) (#46698-250MG, Sigma-Aldrich, Munich, Germany) by oral administration to induce hypothyroidism. The duration of the PTU treatment was selected based on a study by Jena and Bhanja [51]. Then, the animals were sacrificed. The blood serum and uterus were collected and immediately frozen. The tissues were stored at −80 • C until mRNA and protein extraction.

mRNA Isolation and Real-Time PCR
mRNA was isolated using the Total RNA Mini Plus Kit (#036-100; A&A Biotechnology; Gdansk, Poland). The quality and quantity of the mRNA were measured using the NanoDrop 100 (Thermo Fisher Scientific; Waltham, MA, USA). Reverse transcription was performed using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (#K1642; Thermo Fisher Scientific; Waltham, MA, USA). Real-time PCR was performed with the ABI Prism 7900 (Applied Biosystems, Life Technologies, Waltham, MA, USA) sequence detection system using the Maxima SYBR Green/ROX qPCR Master Mix (#K0223; Thermo Fisher Scientific, Waltham, MA, USA). PCRs were performed in 384-well plates. The results for the mRNA transcription were normalized to β-actin (ACTB, internal control). The mRNA levels are shown in arbitrary units. The primers were designed using Primer3web version 4.0.0 (http://primer3.ut.ee; accessed on 23 April 2019), and their sequences are shown in Table 1. For the relative quantification of the mRNA levels, the Miner software was used (http://www.miner.ewindup.info; accessed on 23 April 2019).

Statistical Analyses
Statistical analyses were conducted using GraphPad Prism 7 (GraphPad Software, Inc., CA, USA). All the data are shown as the mean ± SEM, and differences were considered to be significantly different at a 95% confidence level (p < 0.05). Analyses were performed using Student's t-tests. Figure 1 shows the T3, T4, and TSH levels in the blood serum samples in the control group and the experimental group with PTU-induced hypothyroidism. The levels of T3 and T4 were significantly decreased (p < 0.05), whereas the level of TSH was significantly increased (p < 0.05) in the blood serum samples from the experimental group in comparison to the control group. Figure 2 shows the E2 and P4 levels in the blood serum samples in the control group and the experimental group with PTU-induced hypothyroidism. The level of E2 was significantly decreased (p < 0.05), whereas that of P4 was not changed (p > 0.05) in the blood serum samples from the experimental group in comparison to the control group. Figure 1 shows the T3, T4, and TSH levels in the blood serum samples in the contro group and the experimental group with PTU-induced hypothyroidism. The levels of T and T4 were significantly decreased (p < 0.05), whereas the level of TSH was significantl increased (p < 0.05) in the blood serum samples from the experimental group in compar son to the control group. Figure 1. The levels of T3, T4, and TSH in blood serum samples (ng/mL). The data are presented as the mean ± SEM. Asterisks indicate significant differences ((* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001), as determined by Student's ttest. Figure 2 shows the E2 and P4 levels in the blood serum samples in the control grou and the experimental group with PTU-induced hypothyroidism. The level of E2 was sig nificantly decreased (p < 0.05), whereas that of P4 was not changed (p > 0.05) in the bloo serum samples from the experimental group in comparison to the control group. Figure 1. The levels of T3, T4, and TSH in blood serum samples (ng/mL). The data are presented as the mean ± SEM. Asterisks indicate significant differences ((* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001), as determined by Student's t-test.        Figure 4 shows the mRNA and protein expression patterns of prostaglandin endoperoxide synthase 2 PTGS2/PTGS2 (COX-2). The mRNA level of PTGS2 was significantly higher in the group with hypothyroidism than the control group, while the protein  Figure 4 shows the mRNA and protein expression patterns of prostaglandin endoperoxide synthase 2 PTGS2/PTGS2 (COX-2). The mRNA level of PTGS2 was significantly higher in the group with hypothyroidism than the control group, while the protein level of PTGS2 was significantly lower in the group with hypothyroidism. Figure 5 shows the mRNA and protein expression patterns of PGE 2 synthases. The mRNA and protein levels of PTGES-2/PTGES-2 were significantly higher in the experimental group with induced hypothyroidism than the control group (p < 0.05). There was significantly higher PTGES-3 mRNA expression in the group of animals with induced hypothyroidism (p < 0.05), while its protein expression was maintained at the same level in both groups. Figure 6 shows the mRNA and protein expression patterns of the PGE 2 receptors. There was no significant difference in the mRNA expression of PTGER1 between the groups, whereas the expression of the PTGER1 protein was significantly lower in the experimental group with induced hypothyroidism than the control group (p < 0.05). The mRNA and protein expression levels of PTGER2/PTGER2 and PTGER3/PTGER3 show similar patterns. The expression levels were significantly lower in the group of animals with hypothyroidism than the control group of rats (p < 0.05). The mRNA and protein expression levels of PTGER4/PTGER4 were significantly higher in the experimental group in comparison to the control group of rats (p < 0.05). Figure 7 shows the mRNA and protein expression patterns of PGF 2α synthase PGFS/PGFS and the receptor for PGF 2α (PTGFR/PTGFR). The PGFS/PGFS expression pattern was similar for both mRNA and protein. It was significantly lower in the group of animals with hypothyroidism than the control group (p < 0.05). The mRNA and protein expression patterns of the PGF 2α receptor PGFR/PGFR were opposite. The mRNA level was significantly higher, while the protein level was significantly lower, in the group of animals with hypothyroidism than the control group (p < 0.05). Figure 8 shows the mRNA and protein expression patterns of prostacyclin synthase PTGIS/PTGIS and the prostacyclin receptor PTGIR/PTGIR. The mRNA level of PTGIS was significantly higher in the group of rats with induced hypothyroidism than the control group of animals, while there was no difference in the protein level of PTGIS between the groups. In the case of the prostacyclin receptor, the mRNA level of PTGIR was significantly higher in the experimental group of animals than the control group, whereas the protein level of PTGIR was significantly lower in the group of rats with hypothyroidism than the control group (p < 0.05). 11, x PTGES-3 mRNA expression in the group of animals with induced hypothyroid 0.05), while its protein expression was maintained at the same level in both group 6 shows the mRNA and protein expression patterns of the PGE2 receptors. Ther significant difference in the mRNA expression of PTGER1 between the groups, the expression of the PTGER1 protein was significantly lower in the experiment with induced hypothyroidism than the control group (p < 0.05). The mRNA and expression levels of PTGER2/PTGER2 and PTGER3/PTGER3 show similar patte expression levels were significantly lower in the group of animals with hypoth than the control group of rats (p < 0.05). The mRNA and protein expression PTGER4/PTGER4 were significantly higher in the experimental group in comp the control group of rats (p < 0.05). Figure 7 shows the mRNA and protein ex patterns of PGF2α synthase PGFS/PGFS and the receptor for PGF2α (PTGFR/PTG PGFS/PGFS expression pattern was similar for both mRNA and protein. It wa cantly lower in the group of animals with hypothyroidism than the control gr 0.05). The mRNA and protein expression patterns of the PGF2α receptor PGFR/PG opposite. The mRNA level was significantly higher, while the protein level wa cantly lower, in the group of animals with hypothyroidism than the control gr 0.05). Figure 8 shows the mRNA and protein expression patterns of prostacyclin PTGIS/PTGIS and the prostacyclin receptor PTGIR/PTGIR. The mRNA level of PT significantly higher in the group of rats with induced hypothyroidism than th group of animals, while there was no difference in the protein level of PTGIS bet groups. In the case of the prostacyclin receptor, the mRNA level of PTGIR wa cantly higher in the experimental group of animals than the control group, wh protein level of PTGIR was significantly lower in the group of rats with hypoth than the control group (p < 0.05).          Figure 9 shows the concentrations of PGs in the uterine tissue. The concentrations of PGE 2 , PGF 2α , and PGI 2 in the uterine tissue were significantly lower in the experimental group of animals with induced hypothyroidism than the control group of animals (p < 0.05).  Figure 9 shows the concentrations of PGs in the uterine tissue. The concentrations of PGE2, PGF2α, and PGI2 in the uterine tissue were significantly lower in the experimental group of animals with induced hypothyroidism than the control group of animals (p < 0.05).

Discussion
Our present study provides a new understanding of the possible relationship between thyroid hormones and uterine receptivity, as well as prostaglandin signaling pathways in the rat uterus. To the best of our knowledge, this is the first report to investigate the effect of PTU-induced hypothyroidism on uterine receptivity and the uterine synthesis of prostaglandins in rats.
Hypothyroidism is a disease characterized by a defect in the production of thyroid hormones due to the insufficient stimulation by TSH of normal thyroid gland function. This condition is the consequence of an anatomic or functional disorder of the pituitary gland or the hypothalamus, resulting in variable alterations of TSH secretion [52]. Other causes include a congenital absence of the thyroid, radioiodine therapy for hyperthyroidism, surgical thyroidectomy, various drugs that affect thyroid function or cause thyroid inflammation, and a variety of other rarer causes [53]. Congenital hypothyroidism occurs in ~1/4000 infants [54]. The lifetime risk of overt hypothyroidism is around 5%, and this disease is usually preceded by subclinical hypothyroidism, which has an even higher prevalence (estimated to be up to 9%) [52].
It is known from the literature that thyroid hormones affect reproductive capacity and, in particular, play an important role during implantation and the early stages of embryo development. Successful implantation is the result of reciprocal interactions between the implantation-competent blastocyst and the receptive uterus [55]. Thus, thyroid hormones may also influence angiogenesis and immune function in the uterus during implantation. Angiogenesis is crucial for successful implantation, decidualization, and placentation. In the case of immune function, natural killer (NK) cells have emerged as crucial modulators of implantation and placental angiogenesis. It is known that NK cell concentrations are higher in patients with thyroid autoimmunity and recurrent spontaneous abortion or unexplained infertility. This suggests their involvement in the decrease in uterine receptivity and the increase in pregnancy loss. Thyroid hormones have been reported to significantly influence the concentrations of the female reproductive hormones estradiol (E2) and progesterone (P4) [5,11,56]. In the current study, we observed that the administration of PTU in the drinking water decreased the circulating E2 level compared to control. We did not observe any effect on the P4 level. Our results are partially consistent with the results of Kong et al. [5] and Tohei [57], who found that, in rats with hypothyroidism, the levels of both E2 and P4 were decreased. However, Hapon et al. [58]

Discussion
Our present study provides a new understanding of the possible relationship between thyroid hormones and uterine receptivity, as well as prostaglandin signaling pathways in the rat uterus. To the best of our knowledge, this is the first report to investigate the effect of PTU-induced hypothyroidism on uterine receptivity and the uterine synthesis of prostaglandins in rats.
Hypothyroidism is a disease characterized by a defect in the production of thyroid hormones due to the insufficient stimulation by TSH of normal thyroid gland function. This condition is the consequence of an anatomic or functional disorder of the pituitary gland or the hypothalamus, resulting in variable alterations of TSH secretion [52]. Other causes include a congenital absence of the thyroid, radioiodine therapy for hyperthyroidism, surgical thyroidectomy, various drugs that affect thyroid function or cause thyroid inflammation, and a variety of other rarer causes [53]. Congenital hypothyroidism occurs in~1/4000 infants [54]. The lifetime risk of overt hypothyroidism is around 5%, and this disease is usually preceded by subclinical hypothyroidism, which has an even higher prevalence (estimated to be up to 9%) [52].
It is known from the literature that thyroid hormones affect reproductive capacity and, in particular, play an important role during implantation and the early stages of embryo development. Successful implantation is the result of reciprocal interactions between the implantation-competent blastocyst and the receptive uterus [55]. Thus, thyroid hormones may also influence angiogenesis and immune function in the uterus during implantation. Angiogenesis is crucial for successful implantation, decidualization, and placentation. In the case of immune function, natural killer (NK) cells have emerged as crucial modulators of implantation and placental angiogenesis. It is known that NK cell concentrations are higher in patients with thyroid autoimmunity and recurrent spontaneous abortion or unexplained infertility. This suggests their involvement in the decrease in uterine receptivity and the increase in pregnancy loss. Thyroid hormones have been reported to significantly influence the concentrations of the female reproductive hormones estradiol (E2) and progesterone (P4) [5,11,56]. In the current study, we observed that the administration of PTU in the drinking water decreased the circulating E2 level compared to control. We did not observe any effect on the P4 level. Our results are partially consistent with the results of Kong et al. [5] and Tohei [57], who found that, in rats with hypothyroidism, the levels of both E2 and P4 were decreased. However, Hapon et al. [58] found significantly higher luteal P4 content on Day 21 of pregnancy in rats with hypothyroidism compared to control, suggesting that hypothyroidism impairs CL function during gestation, delaying the onset of parturition in the rat. As P4 and E2 are necessary for the maturation and development of oocytes, preparing the endometrium for embryo implantation, and are important in the establishment and maintenance of early pregnancy, changes in the physiological levels of these hormones caused by TH hormones may contribute to disorders of or disrupt the reproductive system. Furthermore, in our experiment, in the groups of animals with PTU-induced hypothyroidism, the levels of homeobox A10 (HOXA10/HOXA10) and osteopontin (OPN/OPN), which are considered to be uterine receptivity markers [13], were significantly lower in comparison to those in the control group. Hoxa10/HOXA10 has been found to be directly involved in embryogenesis and embryo implantation via the regulation of downstream genes. The cyclical endometrial expression of this factor, with a peak of expression occurring during the window of implantation, is observed in response to E2 and P4 [59]. Osteopontin, a member of the extracellular matrix (ECM) protein family, is involved in many physiological and pathological processes, including cell adhesion, cell proliferation and differentiation, angiogenesis, and tumor metastasis. Weintraub et al. [60] showed that OPN-deficient mice manifested a decreased pregnancy rate during mid-gestation and that the knockdown of OPN in mouse endometrial stromal cells restrained trophoblast invasion in vitro. Additionally, OPN was proved to be activated in mouse endometrial stromal cells (mESCs) and human endometrial stromal cells (hESCs) by E2 and P4 [61,62]. These results suggest that OPN plays an important role in regulating blastocyst implantation and decidualization. Our data confirm that, in hypothyroidism, uterine receptivity is reduced. A disturbed E2 level and decreased expression of HOXA10 and OPN may result in difficulties with the implantation of blastocysts and the maintenance of pregnancy.
Prostaglandin endoperoxide synthase 2 (PTGS2) is involved in inflammation and in essential reproductive processes, including ovulation, fertilization, implantation, and decidualization. PTGS2-deficient females are infertile, with abnormalities in ovulation, fertilization, implantation, and decidualization [63,64]. PTGS2 is the key enzyme involved in prostaglandin synthesis [65,66]. Gillio-Meina et al. [42] showed that the PTGS2 protein is gradually expressed in the rat endometrium in response to deciduogenic stimuli. In our study, we showed that the PTGS2 protein level was significantly lower in the group of rats with induced hypothyroidism in comparison to the control group of animals. Combining that information with the knowledge that induced hypothyroidism in rats caused a reduction in the absolute volume of the endometrium [7,8], it may be speculated that the decreased level of PTGS2 in the uterus in the group of rats with induced hypothyroidism was caused by the significantly reduced volume of endometrium.
PTGES-2 and PTGES-3 are responsible for the conversion of PGH 2 to PGE 2 , while PGFS converts PGH 2 to PGF 2α . PGIS is responsible for the conversion of PGH 2 to PGI 2 . These enzymes are involved in the reproductive processes [25,26]. Their expression at implantation sites in rats and other species has been reported [48,49]. In our study, we showed higher expression levels of PTGES-2 in the experimental group of animals, whereas the expression of PTGES-3 and PGIS was at the same level in the experimental and control groups. The expression of PGFS was significantly lower in the group of rats with hypothyroidism. This suggests that hypothyroidism may stimulate the synthesis of PTGES2 and inhibit the synthesis of PGFS but does not affect the synthesis of PTGES-3 or PGIS. In the present study, the mRNA and protein expression patterns of PGFR as well as PGIR were opposite. The mRNA level was significantly higher, while the protein level was significantly lower in the group of animals with hypothyroidism in comparison to the control group. This could be a result of post-translational changes in proteins. These changes regulate the functional activities of proteins in cells and their interactions with other cellular molecules. It is considered that this modification plays a key role in modulating protein function.
It has been reported that the prostaglandin receptors EP1, EP3, and FP promote uterine smooth muscle contraction, while EP2 and EP4 affect uterine smooth muscle relaxation [29,34,35]. Blesson et al. [29] also indicated that the expression levels of the receptors of PGs are regulated by steroid hormones. In the human endometrium, it has been found that the levels of PG receptors vary in a phase-specific manner. EP2, EP3, and EP4 dominate in the mid-secretory phase, while EP1 dominates in the early-secretory phase, and FP dominates in the proliferative phase. This suggests that the expression of those receptors may be regulated by E2 and P4 [29,36]. Our study showed that induced hypothyroidism caused lower expression levels of EP1, EP3, and FP and higher expression levels of EP2 and EP4. It suggests that hypothyroidism leads to the impairment of the uterus' function and disturbances of the estrous cycle by inducing changes in the expression levels of PG receptors in the uterine tissue. Furthermore, EP2, EP3, and EP4 might participate in the regulation of stromal edema, endometrial blood flow, and blood vessel permeability [29,36]. This, in turn, indicates that hypothyroidism can cause abnormalities in the cyclic changes in the uterine blood supply via changes in the expression of those receptors in the rat uterus. It is known that, in rodents, PGI 2 s signaling and the expression of its receptor (PTGIR) are very important components of embryo-uterus interactions and are essential for successful implantation [25,39,41,42]. In mice, PGI 2 is also critical for endometrial decidualization and embryo implantation [25,39]. In addition, PGI 2 increases embryonic cell proliferation and reduces apoptosis [25,44,45]. It also enhances embryo hatching and the live birth potential of mouse embryos [25,46,47]. In our study, we also demonstrated a lower level of PTGIR in the group of animals with induced hypothyroidism. This implies that hypothyroidism may cause changes in endometrial decidualization, leading to difficulties in embryo implantation and the maintenance of pregnancy.

Conclusions
In summary, this study is the first to characterize the influence of hypothyroidism on uterine receptivity and PG signaling in uterine tissue in a rat model. The results show that hypothyroidism impairs uterine receptivity by decreasing the level of E2 as well as the expression of the uterine-receptivity factors HOXA10 and OPN. Hypothyroidism also causes changes in the expression of elements of the PGE 2 , PGF 2α , and PGI 2 signaling pathways, and changes in the concentrations of those prostaglandins in uterine tissue. In general, it decreases the expression of those factors in the rat uterus. It is known that prostaglandins are strongly involved in uterine functions, such as decidualization and blastocyst implantation, and therefore, hypothyroidism causes abnormalities in the female reproductive system. Our results suggest that hypothyroidism may interfere with the prostaglandin signaling pathway, which may further result in a reduction in uterine receptivity. However, this hypothesis requires further investigation.

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author, I.K.-Z.