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Volume 18, Issue 10 (October 2020)
IJRM 2020, 18(10): 815-824 |
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Ajdary M, Zandieh Z, Amjadi F S, Keyhanfar F, Mehdizadeh M, Aflatoonian R. Interaction of sperm with endometrium can regulate genes involved in endometrial receptivity pathway in mice: An experimental study. IJRM 2020; 18 (10) :815-824
URL: http://ijrm.ir/article-1-1474-en.html
URL: http://ijrm.ir/article-1-1474-en.html
Marziyeh Ajdary1 
, Zahra Zandieh2 , Fatemeh Sadat Amjadi2 , Fariborz Keyhanfar3 , Mehdi Mehdizadeh4 , Reza Aflatoonian * 5
, Zahra Zandieh2 , Fatemeh Sadat Amjadi2 , Fariborz Keyhanfar3 , Mehdi Mehdizadeh4 , Reza Aflatoonian * 5
1- Endometriosis Research Center, Iran University of Medical Sciences, Tehran, Iran. Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran.
2- Department of Anatomical Science, Iran University of Medical Sciences, Tehran, Iran.
3- Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
4- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran. Department of Anatomical Science, Iran University of Medical Sciences, Tehran, Iran.
5- Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. ,r.aflatoonian@gmail.com
2- Department of Anatomical Science, Iran University of Medical Sciences, Tehran, Iran.
3- Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
4- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran. Department of Anatomical Science, Iran University of Medical Sciences, Tehran, Iran.
5- Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. ,
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1. Introduction
While several studies have been performed to provide potential molecules, candidates for endometrial receptivity (1-5), there is yet no confirmed evidence about these molecules and the role of sperm in endometrial receptivity. During transportation, fertilization, and development, spermatozoa interact with the endometrial cells and create a category of secretory molecules (6), influencing the endometrial receptivity (7).
In a study, Berger and colleague demonstrated that leukemia inhibitory factor (LIF), Leukemia inhibitory factor receptor (LIFR), Homeobox gene 10 (HOXA10), Mucin 1 (MUC1), Progesterone receptor (PGR), Colony Stimulating Factor (CSF), Vascular endothelial growth factor (VEGF), Heparin-binding EGF-like growth factor (HBEGF), Epidermal growth factor (EGF), and Fibroblast Growth Factor 2 (FGF2) are involved in endometrial receptivity (8). In a study of mice and pigs, Robertson and colleague found that the seminal plasma is effective on the cytokines and epithelial cocaine cells of the uterus, which makes it successful in implantation (9). Several hormones and cytokines that play a role in endometrial receptivity have been established by studies. HOXA10, LIF are involved in the proliferation, differentiation, and decidualization of the endometrium and are highly expressed at this phase (10). While the LIF-R gene plays a role in mediating the action of the leukemia-inhibitory factor that finally causes a proliferation (11), HB-EGF plays an endometrial preparation for embryo acceptance and is expressed in mice on the fourth day of gestation (12). VEGF, FGF-2, EGF are involved in angiogenesis that is important at the time of implantation and transportation of nutrients and oxygen to the embryo (13). MUC1 at the surface of uterine epithelia, acts as a barrier to microbial infection and enzymatic attack. At the time of implantation, the expression of MUC1 reduce PGR and PGR and MUC1 have different expression during a natural cycle in mice. MUC1 and PGR are upregulated at estrous and at early pregnancy for four days and reduced on day 4 of the pregnancy (14). Additionally, CSF plays an important role in the proliferation and differentiation, and starting from the third day of gestation, its expression increases (15).
In this study, the effects of sperm interaction with endometrium on endometrial receptivity gene expression (LIF, LIFR, HOXA10, MUC1, PGR, CSF, VEGF, HBEGF, EGF, and FGF2) in a mouse model (in-vivo) were investigated. All of these genes fall in the path of implantation and affect the endometrial readiness in embryo acceptance. Because studies have shown that both spermatozoa and semen are effective on pinopods (1, 16), in this study, we sought to investigate the effect of sperm on implantation and the genes important for uterine reception.
2. Materials and Methods
2.1. Materials and equipment
Entire objective includes Ketamine (Alfasan, Woerden- Holland), Xylazine (Alfasan, Woerden-Holland), TRIzol reagent (Sigma, Pool, UK), cDNA Synthesis Kit (Invitrogen, Paisley, UK), primers (Roche, Basel, Switzerland), and SYBR Green PCR Master Mix (Invitrogen, Paisley, UK). Entire Apparatus includes an inverted microscope (Nikon UK, Ltd.), Nanodrop ND-100 spectrophotometer (Thermo Fisher Scientific, USA), thermocycler-T100 (Bio-Rad, USA), Opticon II system (Invitrogen, Paisley, UK), Step One Plus Real-time PCR (Applied Biosystems, Foster, USA).
2.2. Animal preparation
In this experimental study, 30 female and 10 male NMRI mice weighing 20-50 gr at 8 wk of age were used. Half of the male animals were subjected to vasectomy and anesthetized with Ketamine (10 mg/kg) or Xylazine (2%). One month after wound healing, the mice were selected to continue the experiments.
While several studies have been performed to provide potential molecules, candidates for endometrial receptivity (1-5), there is yet no confirmed evidence about these molecules and the role of sperm in endometrial receptivity. During transportation, fertilization, and development, spermatozoa interact with the endometrial cells and create a category of secretory molecules (6), influencing the endometrial receptivity (7).
In a study, Berger and colleague demonstrated that leukemia inhibitory factor (LIF), Leukemia inhibitory factor receptor (LIFR), Homeobox gene 10 (HOXA10), Mucin 1 (MUC1), Progesterone receptor (PGR), Colony Stimulating Factor (CSF), Vascular endothelial growth factor (VEGF), Heparin-binding EGF-like growth factor (HBEGF), Epidermal growth factor (EGF), and Fibroblast Growth Factor 2 (FGF2) are involved in endometrial receptivity (8). In a study of mice and pigs, Robertson and colleague found that the seminal plasma is effective on the cytokines and epithelial cocaine cells of the uterus, which makes it successful in implantation (9). Several hormones and cytokines that play a role in endometrial receptivity have been established by studies. HOXA10, LIF are involved in the proliferation, differentiation, and decidualization of the endometrium and are highly expressed at this phase (10). While the LIF-R gene plays a role in mediating the action of the leukemia-inhibitory factor that finally causes a proliferation (11), HB-EGF plays an endometrial preparation for embryo acceptance and is expressed in mice on the fourth day of gestation (12). VEGF, FGF-2, EGF are involved in angiogenesis that is important at the time of implantation and transportation of nutrients and oxygen to the embryo (13). MUC1 at the surface of uterine epithelia, acts as a barrier to microbial infection and enzymatic attack. At the time of implantation, the expression of MUC1 reduce PGR and PGR and MUC1 have different expression during a natural cycle in mice. MUC1 and PGR are upregulated at estrous and at early pregnancy for four days and reduced on day 4 of the pregnancy (14). Additionally, CSF plays an important role in the proliferation and differentiation, and starting from the third day of gestation, its expression increases (15).
In this study, the effects of sperm interaction with endometrium on endometrial receptivity gene expression (LIF, LIFR, HOXA10, MUC1, PGR, CSF, VEGF, HBEGF, EGF, and FGF2) in a mouse model (in-vivo) were investigated. All of these genes fall in the path of implantation and affect the endometrial readiness in embryo acceptance. Because studies have shown that both spermatozoa and semen are effective on pinopods (1, 16), in this study, we sought to investigate the effect of sperm on implantation and the genes important for uterine reception.
2. Materials and Methods
2.1. Materials and equipment
Entire objective includes Ketamine (Alfasan, Woerden- Holland), Xylazine (Alfasan, Woerden-Holland), TRIzol reagent (Sigma, Pool, UK), cDNA Synthesis Kit (Invitrogen, Paisley, UK), primers (Roche, Basel, Switzerland), and SYBR Green PCR Master Mix (Invitrogen, Paisley, UK). Entire Apparatus includes an inverted microscope (Nikon UK, Ltd.), Nanodrop ND-100 spectrophotometer (Thermo Fisher Scientific, USA), thermocycler-T100 (Bio-Rad, USA), Opticon II system (Invitrogen, Paisley, UK), Step One Plus Real-time PCR (Applied Biosystems, Foster, USA).
2.2. Animal preparation
In this experimental study, 30 female and 10 male NMRI mice weighing 20-50 gr at 8 wk of age were used. Half of the male animals were subjected to vasectomy and anesthetized with Ketamine (10 mg/kg) or Xylazine (2%). One month after wound healing, the mice were selected to continue the experiments.
- Fifteen female mice were mated with the vasectomized male mice. Two days later, they were coupled with a sexually mature male mouse at 05:00 in the afternoon. The next morning, the vaginal plugs were examined, and therefore, this day was named the first gestation day. Female mice were sacrificed with perfusion-fixation technique on day 1.5 post coitus. The uterus was removed and the endometrial tissues were collected and frozen in liquid nitrogen and stored at -80ºC for examination by quantitative real-time PCR (Q-PCR).
- Fifteen female mice were mated with the non-vasectomized male mice. Two days later, they were coupled with a sexually mature male mouse at 05:00 in the afternoon two days later. The next morning, the vaginal plugs were examined and therefore, this day was named the first gestation day. The female mice were then sacrificed with the perfusion-fixation technique on day 1.5 post copulation. The endometrial tissue on day 1.5 was washed by PBS and sperms present in the uterus were observed under a microscope (1).
The uterus was removed, washed, and the endometrial tissues were collected and frozen in liquid nitrogen and stored at -80ºC for examination under Q-PCR.
2.3. RNA extraction and real-time quantitative reverse-transcription PCR
Extraction and purification of the total RNAs from the endometrial tissue were performed by TRIzol reagent (Sigma, Pool, UK) based on the given instructions. A Nanodrop ND-100 spectrophotometer was applied to determine the exact concentration of RNA. In order to evaluate the level of gene expression, a Q-PCR was used against HBEGF, LIF, PGR, FGF2, VEGF, HOXA10, LIFR, CSF, EGF, and MUC1. A transcript or High-Fidelity cDNA Synthesis Kit (Invitrogen, Paisley, UK) was used for the reverse transcription of a 500-ng sample of RNA using oligo (dT) primers (Roche, Basel, Switzerland). In order to amplify the cDNA (1 μL), the SYBR Green PCR Master Mix (Invitrogen, Paisley, UK) and the Opticon II system (Invitrogen, Paisley, UK) were used according to the instructions. The annealing temperature of 60ºC for all tested genes were done to find the 40 PCR cycles. Further, primers were specifically designed between two adjacent exons (Gene Runner program). Table I presents all the used sequences. The concentrations of mRNA for these genes (Ct) were regulated via the reference gene glyceraldehyde-3-phosphate dehydrogenase (β-actin) through subtracting the Ct value of β-actin from the Ct value of the sample (ΔCt = CtSample - CtReference). 2-ΔΔCt was applied for quantification of the relative expression of the target gene to the calibrator (17, 18).
Additionally, the Primer Premier 5.0 software was used to design primer pairs specified for each gene. The applied primers for real-time PCR are presented in Table I.
2.4. Ethical consideration
All experiments were done following the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Iran University of Medical Sciences) and were approved by the Research and Ethics Committee of Iran University of Medical Sciences (code: IR-IUMS.1394.94-05-117-27524).
2.5. Statistical Analysis
All groups were analyzed using an independent sample t test. Statistical analyses were performed using the SPSS (version 21.0, IBM, New York, USA) software. The results were considered significant at p < 0.05 and are expressed as Mean ± SD.
3. Results
3.1. Gene expression in endometrial tissue
The mRNA levels of LIF, MUC1, VEGF, EFG and FGF2 were all significantly higher in the group that was mated with the non-vasectomized males compared with the group mated with the vasectomized males (p < 0.05) (Figure 1, Table II).
No significant differences were found in the expression of PGR, CSF, HBEGF, and HOXA10 mRNA between the group that was mated with non-vasectomized males and the group that was mated with vasectomized males; however, p > 0.05 was observed (Figure 1, Table II).
The LIF, LIFR, MUC1, and FGF2 were mainly expressed in the cytoplasm of both the endometrial luminal epithelial cells and the glandular epithelial cells (Figure 1). As shown in Figure 1, the expression levels of LIF, LIFR, MUC1, and FGF2 in the group that was mated with the non-vasectomized males were higher compared with the group that was mated with the vasectomized males (all p < 0.05). As shown in Figure 1, the expression levels of HOXA10 in the group that was mated with the non-vasectomized males were lower compared with the group that was mated with the vasectomized males.
4. Discussion
The results of this study confirmed that seminal plasma regulates the LIF, LIFR, HOXA10, MUC1, PGR, CSF, VEGF, HBEGF, EGF, and FGF2 expression in the mouse endometrium in vivo. According to our results, it can be recommended that seminal plasma, by changing the gene expression, can regulate the environment within the endometrium.
During the interaction of seminal plasma-endometrium, genes involved in proliferation, differentiation, and decidualization are expressed by the epithelial cell, and endometrial cells secrete inflammatory factors, growth factor, cytokines, including LIF, VEGF, FGF2, and EGF (19-21). The VEGF, FGF2, and EGF increase endometrial angiogenesis and subsequently lead to a successful implantation (22). In addition, the expression of the HBEGF factor at this stage prepares the uterus for endometrial receptivity (23). During the interaction of the seminal plasma-endometrium, the expression of HBEGF, CSF, PGR increases, which plays an important role in the proliferation and differentiation (15). At one stage of estrus (the time of mating of the animal), the innate immune factors in the uterus of the female animal increase; one of the most important of these factors is the MUC1 gene, which prevents microbes from entering the uterus (24).
Intriguingly, numerous in vitro studies have investigated the effects of seminal plasma on endometrium, however, the importance of this study compared to the in vitro investigations, is that the in vitro studies ignore the effect of female hormones on sperm and endometrial interaction (19), (25-27). The results of various studies have shown that sperm-epithelial cell interaction creates a successful embryo implantation in three different ways.
The first theory is to achieve a successful fertilization. Sperm should be capacitated in the female reproductive tract, necessitating a sperm interaction with the female epithelial cells (28). The first prerequisite for pregnancy in humans and other mammals is fertilization; to achieve this, the sperm capacitation in the female reproductive tract is completed, whereas this capacitation is initiated by the interaction of the sperm with the cervix (29). A study conducted by Reeve and Ledger showed that the Arg-Gly-Asp (RGD) sequence in the epithelial cell of endometrium makes a better interaction between the sperm and the endometrium (30). This interaction can be one of the important factors in the embryo implantation (31). The movement of the sperm in the uterine epithelial cell leads to the expression of some factors from secretory cells that provide the conditions and nutrients for embryo implantation (32), sperm-endometrium interaction cause the ATP secretion from the epithelial cell of the endometrium, an essential factor for the capacitation and the movement of the cell to the fallopian tube
(33).
The second theory is that an interaction between sperm and the endometrium leads to the expression of inflammatory factors involved in the implantation pathway (32, 34). Kaczmarek and colleague showed that seminal plasma is effective in expressing endometrial cytokines in rabbits, and had been led to the expression of EGF, VEGF, and FGF2 (35). In addition, Gutsche and colleague confirmed that seminal plasma induces the expression of cytokines and growth factors such as LIF and VEGF in the endometrium (36). Previous studies have shown that the passage of seminal plasma from endometrium lead to genomic alteration, such as the expression of FGF (37), which is consistent with the present study. Carp and colleague showed a higher implantation rate in mice exposed to semen after embryo transfer in their endometrium (38). The VEGF, FGF2, and EGF increase endometrial angiogenesis and subsequently lead to a successful implantation (22). The LIF is also one of the cytokines that plays a role in the pathway of proliferation, differentiation, and decidualization (21). Artificial insemination in sheep also benefits from cervical spermatozoa exposure and results in an increased percentage of pregnant ewes (39). As shown in Bellinge and colleague’s study, exposing the uterus to sperm during the oocyte retrieval in an IVF cycle increases the implantation rate (40). However, clinical studies have shown that exposure to semen in ART during embryo transfer increases the embryo implantation rate, but the mechanism of this effect is yet unknown.
The third theory is that the interaction between seminal plasma and the endometrium is effective on steroid hormones, prostaglandins, and peptide hormones (41). As mentioned earlier, the sperm-endometrium interaction causes the secretion of inflammatory factors that affect the steroid hormones and prostaglandins (42). Steroid hormones play a role in the secretion of inflammatory factors involved in innate immunity, including cytokines (43). Cytokines play an important role in endometrial receptivity, which is consistent with the second theory. In this study, the expression of genes involved in the pathway of cytokines such as LIF was increased.
Moreover, studies about the sperm and seminal plasma-endometrium interaction are not sufficient and this is an open issue. Therefore, we tried to provide information about the potential effects of sperm and seminal plasma-uterine epithelium interaction in the genes involved in endometrial receptivity-related pathways. The results of this study have shown its use in IVF. The study also examines the human cellular level, determines the appropriate dose and volume of sperm seminal fluid to influence embryo implantation, and can be used for uterine preparation in IVF cycles.
According to the results of the present study, it can be concluded that seminal plasma regulates the LIF, LIFR, HOXA10, MUC1, PGR, CSF, VEGF, HBEGF, EGF, and FGF2 expression in the mouse endometrium in vivo model, and because these genes play an important role in implantation and can increase the rate of implantation by facilitating the receptivity of the endometrium. It should be noted that in patients undergoing IVF cycles, the seminal plasma cannot be contacted with endometrium and patients will avoid intercourse before and after taking the ovum. It can be considered that implantation failures in these patients can be attributed to the lack of the stimulation of endometrial cells from seminal plasma.
5. Conclusion
Finally, it was found that the seminal plasma is effective in expressing the involved genes in implantation in the in vivo model. In fact, seminal plasma increases endometrial receptivity prior to an embryo transfer. Seminal plasma induces the expression of LIF, LIFR, MUC1, VEGF, EGF, and FGF2 genes that increase stromal cell survival and endometrial tissue proliferation before an implantation. According to the obtained results in this study, seminal plasma can be introduced as an auxiliary and effective factor in successful implantation. Taken together, our findings and those of others suggest that the effects of seminal plasma on the endometrium and pregnancy should be investigated in an in vivo/in vitro model for confirmation of these results.
Acknowledgments
The authors gratefully acknowledge the financial support of the Cellular and Molecular Research Center of the Iran University of Medical Sciences (94-05-117-27524) and the Iran National Science Foundation (INSF) for funding this work through a grant (95849005).
Conflict of Interest
The authors declare that there is no competing interest.
2.3. RNA extraction and real-time quantitative reverse-transcription PCR
Extraction and purification of the total RNAs from the endometrial tissue were performed by TRIzol reagent (Sigma, Pool, UK) based on the given instructions. A Nanodrop ND-100 spectrophotometer was applied to determine the exact concentration of RNA. In order to evaluate the level of gene expression, a Q-PCR was used against HBEGF, LIF, PGR, FGF2, VEGF, HOXA10, LIFR, CSF, EGF, and MUC1. A transcript or High-Fidelity cDNA Synthesis Kit (Invitrogen, Paisley, UK) was used for the reverse transcription of a 500-ng sample of RNA using oligo (dT) primers (Roche, Basel, Switzerland). In order to amplify the cDNA (1 μL), the SYBR Green PCR Master Mix (Invitrogen, Paisley, UK) and the Opticon II system (Invitrogen, Paisley, UK) were used according to the instructions. The annealing temperature of 60ºC for all tested genes were done to find the 40 PCR cycles. Further, primers were specifically designed between two adjacent exons (Gene Runner program). Table I presents all the used sequences. The concentrations of mRNA for these genes (Ct) were regulated via the reference gene glyceraldehyde-3-phosphate dehydrogenase (β-actin) through subtracting the Ct value of β-actin from the Ct value of the sample (ΔCt = CtSample - CtReference). 2-ΔΔCt was applied for quantification of the relative expression of the target gene to the calibrator (17, 18).
Additionally, the Primer Premier 5.0 software was used to design primer pairs specified for each gene. The applied primers for real-time PCR are presented in Table I.
2.4. Ethical consideration
All experiments were done following the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Iran University of Medical Sciences) and were approved by the Research and Ethics Committee of Iran University of Medical Sciences (code: IR-IUMS.1394.94-05-117-27524).
2.5. Statistical Analysis
All groups were analyzed using an independent sample t test. Statistical analyses were performed using the SPSS (version 21.0, IBM, New York, USA) software. The results were considered significant at p < 0.05 and are expressed as Mean ± SD.
3. Results
3.1. Gene expression in endometrial tissue
The mRNA levels of LIF, MUC1, VEGF, EFG and FGF2 were all significantly higher in the group that was mated with the non-vasectomized males compared with the group mated with the vasectomized males (p < 0.05) (Figure 1, Table II).
No significant differences were found in the expression of PGR, CSF, HBEGF, and HOXA10 mRNA between the group that was mated with non-vasectomized males and the group that was mated with vasectomized males; however, p > 0.05 was observed (Figure 1, Table II).
The LIF, LIFR, MUC1, and FGF2 were mainly expressed in the cytoplasm of both the endometrial luminal epithelial cells and the glandular epithelial cells (Figure 1). As shown in Figure 1, the expression levels of LIF, LIFR, MUC1, and FGF2 in the group that was mated with the non-vasectomized males were higher compared with the group that was mated with the vasectomized males (all p < 0.05). As shown in Figure 1, the expression levels of HOXA10 in the group that was mated with the non-vasectomized males were lower compared with the group that was mated with the vasectomized males.
4. Discussion
The results of this study confirmed that seminal plasma regulates the LIF, LIFR, HOXA10, MUC1, PGR, CSF, VEGF, HBEGF, EGF, and FGF2 expression in the mouse endometrium in vivo. According to our results, it can be recommended that seminal plasma, by changing the gene expression, can regulate the environment within the endometrium.
During the interaction of seminal plasma-endometrium, genes involved in proliferation, differentiation, and decidualization are expressed by the epithelial cell, and endometrial cells secrete inflammatory factors, growth factor, cytokines, including LIF, VEGF, FGF2, and EGF (19-21). The VEGF, FGF2, and EGF increase endometrial angiogenesis and subsequently lead to a successful implantation (22). In addition, the expression of the HBEGF factor at this stage prepares the uterus for endometrial receptivity (23). During the interaction of the seminal plasma-endometrium, the expression of HBEGF, CSF, PGR increases, which plays an important role in the proliferation and differentiation (15). At one stage of estrus (the time of mating of the animal), the innate immune factors in the uterus of the female animal increase; one of the most important of these factors is the MUC1 gene, which prevents microbes from entering the uterus (24).
Intriguingly, numerous in vitro studies have investigated the effects of seminal plasma on endometrium, however, the importance of this study compared to the in vitro investigations, is that the in vitro studies ignore the effect of female hormones on sperm and endometrial interaction (19), (25-27). The results of various studies have shown that sperm-epithelial cell interaction creates a successful embryo implantation in three different ways.
The first theory is to achieve a successful fertilization. Sperm should be capacitated in the female reproductive tract, necessitating a sperm interaction with the female epithelial cells (28). The first prerequisite for pregnancy in humans and other mammals is fertilization; to achieve this, the sperm capacitation in the female reproductive tract is completed, whereas this capacitation is initiated by the interaction of the sperm with the cervix (29). A study conducted by Reeve and Ledger showed that the Arg-Gly-Asp (RGD) sequence in the epithelial cell of endometrium makes a better interaction between the sperm and the endometrium (30). This interaction can be one of the important factors in the embryo implantation (31). The movement of the sperm in the uterine epithelial cell leads to the expression of some factors from secretory cells that provide the conditions and nutrients for embryo implantation (32), sperm-endometrium interaction cause the ATP secretion from the epithelial cell of the endometrium, an essential factor for the capacitation and the movement of the cell to the fallopian tube
(33).
The second theory is that an interaction between sperm and the endometrium leads to the expression of inflammatory factors involved in the implantation pathway (32, 34). Kaczmarek and colleague showed that seminal plasma is effective in expressing endometrial cytokines in rabbits, and had been led to the expression of EGF, VEGF, and FGF2 (35). In addition, Gutsche and colleague confirmed that seminal plasma induces the expression of cytokines and growth factors such as LIF and VEGF in the endometrium (36). Previous studies have shown that the passage of seminal plasma from endometrium lead to genomic alteration, such as the expression of FGF (37), which is consistent with the present study. Carp and colleague showed a higher implantation rate in mice exposed to semen after embryo transfer in their endometrium (38). The VEGF, FGF2, and EGF increase endometrial angiogenesis and subsequently lead to a successful implantation (22). The LIF is also one of the cytokines that plays a role in the pathway of proliferation, differentiation, and decidualization (21). Artificial insemination in sheep also benefits from cervical spermatozoa exposure and results in an increased percentage of pregnant ewes (39). As shown in Bellinge and colleague’s study, exposing the uterus to sperm during the oocyte retrieval in an IVF cycle increases the implantation rate (40). However, clinical studies have shown that exposure to semen in ART during embryo transfer increases the embryo implantation rate, but the mechanism of this effect is yet unknown.
The third theory is that the interaction between seminal plasma and the endometrium is effective on steroid hormones, prostaglandins, and peptide hormones (41). As mentioned earlier, the sperm-endometrium interaction causes the secretion of inflammatory factors that affect the steroid hormones and prostaglandins (42). Steroid hormones play a role in the secretion of inflammatory factors involved in innate immunity, including cytokines (43). Cytokines play an important role in endometrial receptivity, which is consistent with the second theory. In this study, the expression of genes involved in the pathway of cytokines such as LIF was increased.
Moreover, studies about the sperm and seminal plasma-endometrium interaction are not sufficient and this is an open issue. Therefore, we tried to provide information about the potential effects of sperm and seminal plasma-uterine epithelium interaction in the genes involved in endometrial receptivity-related pathways. The results of this study have shown its use in IVF. The study also examines the human cellular level, determines the appropriate dose and volume of sperm seminal fluid to influence embryo implantation, and can be used for uterine preparation in IVF cycles.
According to the results of the present study, it can be concluded that seminal plasma regulates the LIF, LIFR, HOXA10, MUC1, PGR, CSF, VEGF, HBEGF, EGF, and FGF2 expression in the mouse endometrium in vivo model, and because these genes play an important role in implantation and can increase the rate of implantation by facilitating the receptivity of the endometrium. It should be noted that in patients undergoing IVF cycles, the seminal plasma cannot be contacted with endometrium and patients will avoid intercourse before and after taking the ovum. It can be considered that implantation failures in these patients can be attributed to the lack of the stimulation of endometrial cells from seminal plasma.
5. Conclusion
Finally, it was found that the seminal plasma is effective in expressing the involved genes in implantation in the in vivo model. In fact, seminal plasma increases endometrial receptivity prior to an embryo transfer. Seminal plasma induces the expression of LIF, LIFR, MUC1, VEGF, EGF, and FGF2 genes that increase stromal cell survival and endometrial tissue proliferation before an implantation. According to the obtained results in this study, seminal plasma can be introduced as an auxiliary and effective factor in successful implantation. Taken together, our findings and those of others suggest that the effects of seminal plasma on the endometrium and pregnancy should be investigated in an in vivo/in vitro model for confirmation of these results.
Acknowledgments
The authors gratefully acknowledge the financial support of the Cellular and Molecular Research Center of the Iran University of Medical Sciences (94-05-117-27524) and the Iran National Science Foundation (INSF) for funding this work through a grant (95849005).
Conflict of Interest
The authors declare that there is no competing interest.
Type of Study: Original Article |
Subject:
Reproductive Biology
References
1. 1. Özatika O, Mungan T, Dag I, Musmuld A. The effect of sperm activation on pinopod formation in endometrial epithelium. J Anat Soc India 2016; 65: S5-S10.
2. 2. Rincón A, Bolumar D, Valbuena D, Simón C. Use of Molecular markers of endometrial receptivity. In: Rizk B, Khalaf Y. Controversies in assisted reproduction. USA: CRC Press; 2020.
3. 3. Neykova K, Tosto V, Giardina I, Tsibizova V, Vakrilov G. Endometrial receptivity and pregnancy outcome. J Matern Fetal Neonatal Med 2020; 2: 1-15.
4. 4. Ajdary M, Keyhanfar F, Aflatoonian R, Amani A, Amjadi F, Zandieh Z, et al. Design and evaluation of a novel nanodrug delivery system for reducing the side effects of clomiphene citrate on endometrium. DARU J Pharm Sci 2020; 28: 1-10.
5. 5. Ajdary M, Farzan S, Razavi Y, Arabdolatabadi A, Haghparast A. Effects of morphine on serum reproductive hormone levels and the expression of genes involved in fertility-related pathways in male rats. Iran J Pharm Res 2019; 19: 1-26.
6. 6. Lee SK, Kim CJ, Kim DJ, Kang JH. Immune cells in the female reproductive tract. Immune Netw 2015; 15: 16-26.
7. 7. Ibrahim LA, Rizo JA, Fontes PLP, Lamb GC, Bromfield JJ. Seminal plasma modulates expression of endometrial inflammatory meditators in the bovine. Biol Reprod 2019; 100: 660-671.
8. 8. Berger C, Boggavarapu NR, Menezes J, Lalitkumar PGL, Gemzell-Danielsson K. Effects of ulipristal acetate on human embryo attachment and endometrial cell gene expression in an in vitro co-culture system. Hum Reprod 2015; 30: 800-811.
9. 9. Robertson SA. Seminal plasma and male factor signalling in the female reproductive tract. Cell Tissue Res 2005; 322: 43-52.
10. 10. He B, Ni Zl, Kong SB, Lu JH, Wang HB. Homeobox genes for embryo implantation: From mouse to human. Anim Model Exp Med 2018; 1: 14-22.
11. 11. Namiki T, Ito J, Kashiwazaki N. Molecular mechanisms of embryonic implantation in mammals: Lessons from the gene manipulation of mice. Reprod Med Biol 2018; 17: 331-342.
12. 12. Yuan J, Deng W, Cha J, Sun X, Borg JP, Dey SK. Tridimensional visualization reveals direct communication between the embryo and glands critical for implantation. Nat Commun 2018; 9: 603-615.
13. 13. Ribatti D, Tamma R. The chick embryo chorioallantoic membrane as an in vivo experimental model to study human neuroblastoma. J Cell Physiol 2018; 234: 152-157.
14. 14. Fermin LM, Pain SJ, Morel PCH, Gedye KR, Kenyon PR, Blair HT. Effect of exogenous progesterone on embryo size and ewe uterine gene expression in an ovine ‘dam size’model of maternal constraint. Reprod Fertil Dev 2018; 30: 766-778.
15. 15. Camargo-Díaz F, García V, Ocampo-Bárcenas A, González-Marquez H, López-Bayghen E. Colony stimulating factor-1 and leukemia inhibitor factor expression from current-cycle cannula isolated endometrial cells are associated with increased endometrial receptivity and pregnancy. BMC Women's Health 2017; 17: 63-69.
16. 16. Miah AG, Salma U, Hamano K, Schellander K. Physiological roles of relaxin in prefertilizing activities of spermatozoa. Anim Reprod Sci 2015; 161: 1-15.
17. 17. Artimani T, Karimi J, Mehdizadeh M, Yavangi M, Khanlarzadeh E, Ghorbani M, et al. Evaluation of pro-oxidant-antioxidant balance (PAB) and its association with inflammatory cytokines in polycystic ovary syndrome (PCOS). Gynecol Endocrinol 2018; 34: 148-152.
18. 18. Chelongar R, Hajihosseinlo A, Ajdary M. The effect of Igf-1 and pit-1 genes polymorphisms on fat-tail measurements (fat-tail dimensions) in Makooei sheep. Adv Environ Biol 2014; 8: 862-868.
19. 19. Robertson SA, Ingman WV, O'Leary S, Sharkey DJ, Tremellen KP. Transforming growth factor β-a mediator of immune deviation in seminal plasma. J Reprod Immunol 2002; 57: 109-128.
20. 20. Cheng J, Rosario G, Cohen TV, Hu J, Stewart CL. Tissue-specific ablation of the LIF receptor in the murine uterine epithelium results in implantation failure. Endocrinology 2017; 158: 1916-1928.
21. 21. Chen J, Zhao X, Ao L, Yin T, Yang J. Transcriptomic changes and potential regulatory mechanism of intrauterine human chorionic gonadotropin co-cultured with peripheral blood mononuclear cells infusion in mice with embryonic implantation dysfunction. Ann Transl Med 2020; 8: 99-113.
22. 22. Shim SH, Kim JO, Jeon YJ, An HJ, Lee HA, Kim JH, et al. Association between vascular endothelial growth factor promoter polymorphisms and the risk of recurrent implantation failure. Exp Ther Med 2018; 15: 2109-2119.
23. 23. Yue L, Yu HF, Yang ZQ, Tian XC, Zheng LW, Guo B. Egr2 mediates the differentiation of mouse uterine stromal cells responsiveness to HB‐EGF during decidualization. J Exp Zoo B Mol Dev Evol 2018; 330: 215-224.
24. 24. Hu M, Zhang Y, Feng J, Xu X, Zhang J, Zhao W, et al. Uterine progesterone signaling is a target for metformin therapy in PCOS-like rats. J Endocrinol 2018; 237: 123-137.
25. 25. Altmäe S, Martinez-Conejero JA, Salumets A, Simon C, Horcajadas JA, Stavreus-Evers A. Endometrial gene expression analysis at the time of embryo implantation in women with unexplained infertility. Mol Hum Reprod 2010; 16: 178-187.
26. 26. Hamlett WC, Musick JA, Hysell CK, Sever DM. Uterine epithelial‐sperm interaction, endometrial cycle and sperm storage in the terminal zone of the oviducal gland in the placental smoothhound, Mustelus canis. J Exp Zool 2002; 292: 129-144.
27. 27. Zandieh Z, Ashrafi M, Aflatoonian K, Aflatoonian R. Human sperm DNA damage has an effect on immunological interaction between spermatozoa and fallopian tube. Andrology 2019; 7: 228-234.
28. 28. Robertson SA, O'Leary S, Armstrong DT. Influence of semen on inflammatory modulators of embryo implantation. Soc Reprod Fertil Suppl 2006; 62: 231-245.
29. 29. Elweza AE, Ezz MA, Acosta TJ, Talukder AK, Shimizu T, Hayakawa H, et al. A proinflammatory response of bovine endometrial epithelial cells to active sperm in vitro. Mol Reprod Dev 2018; 85: 215-226.
30. 30. Reeve L, Ledger WL, Pacey AA. Does the Arg‐Gly‐Asp (RGD) adhesion sequence play a role in mediating sperm interaction with the human endosalpinx? Hum Reprod 2003; 18: 1461-1468.
31. 31. Gomes GM, Crespilho AM, Leão KM, Jacob JCF, Gomes LPM, Segabinazzi LG, et al. Can sperm selection, inseminating dose, and artificial insemination technique influence endometrial inflammatory response in mares? J Equine Vet Sci 2019; 73: 43-47.
32. 32. Bahar L, Kahraman S, Akkuş M, Baykal T. Fine structure and immunohistochemical evaluation of endometrium in fertile and infertile women with implantation failure. Dicle Tıp Dergisi 2012; 39: 269-275.
33. 33. Burnstock G. Purinergic signalling in the reproductive system in health and disease. Purinergic Signal 2014; 10: 157-187.
34. 34. Cakmak H, Taylor HS. Implantation failure: molecular mechanisms and clinical treatment. Hum Reprod Update 2011; 17: 242-253.
35. 35. Kaczmarek MM, Krawczynski K, Blitek A, Kiewisz J, Schams D, Ziecik AJ. Seminal plasma affects prostaglandin synthesis in the porcine oviduct. Theriogenology 2010; 74: 1207-1220.
36. 36. Gutsche S, Von Wolff M, Strowitzki T, Thaler CJ. Seminal plasma induces mRNA expression of IL-1beta, IL-6 and LIF in endometrial epithelial cells in vitro. Mol Hum Reprod 2003; 9: 785-791.
37. 37. Alvarez-Rodriguez M, Atikuzzaman M, Venhoranta H, Wright D, Rodriguez-Martinez H. Expression of immune regulatory genes in the porcine internal genital tract is differentially triggered by spermatozoa and seminal plasma. Int J Mol Sci 2019; 20: 513-532.
38. 38. Carp HJ, Serr DM, Mashiach S, Nebel L. Influence of insemination on the implantation of transferred rat blastocysts. Gynecol Obstet Invest 1984; 18: 194-198.
39. 39. Maxwell WM, Evans G, Mortimer ST, Gillan L, Gellatly ES, McPhie CA. Normal fertility in ewes after cervical insemination with frozen-thawed spermatozoa supplemented with seminal plasma. Reprod Fertil Dev 1999; 11: 123-126.
40. 40. Bellinge BS, Copeland CM, Thomas TD, Mazzucchelli RE, O’Neil G, Cohen MJ. The influence of patient insemination on the implantation rate in an in vitro fertilization and embryo transfer program. Fertil Steril 1986; 46: 252-256.
41. 41. Cicinelli E, De Ziegler D. Transvaginal progesterone: evidence for a new functional'portal system'flowing from the vagina to the uterus. Hum Reprod Update 1999; 5: 365-372.
42. 42. Pelzer ES, Huygens F, Beagley KW. Steroid hormone dependent inflammation and regulation in the endometrium in women with dysfunctional menstrual cycles: Is there a role for toll-like receptor activation via PAMPs and DAMPs? J Microb Biochem Technol 2016; 8: 344-357.
43. 43. Wira CR, Fahey JV, Sentman CL, Pioli PA, Shen L. Innate and adaptive immunity in female genital tract: cellular responses and interactions. Immunol Rev 2005; 206: 306-335.
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