Interaction of sperm with endometrium can regulate genes involved in endometrial receptivity pathway in mice: An experimental study

Abstract Background Many researchers consider implantation and endometrial receptivity as pertinent issues in reproductive science. Although, several experiments have been performed and their results evaluated, yet there is no confirmed evidence about the related factors and the role of sperm in endometrial receptivity. Objective To investigate the effect of the sperm-endometrium interaction in regulating genes involved in the endometrial receptivity pathway. Materials and Methods In this experimental study, 10 male and 30 female NMRI mice were included, and half of the male cases were vasectomized. The subjects were divided into two groups as follows; group 1 (case) comprised of 15 females mated with 5 non-vasectomized male mice, while group 2 (control) consisted of 15 females mated with 5 vasectomized males. Cases were sacrificed and assessed after 36 hr and the endometrial tissue was extracted and kept at -80°C until the next use. The expression of the endometrial receptivity pathway genes, including VEGF, HBEGF, FGF2, EGF, LIF, LIFR, HOXA10, MUC1, PGR, and CSF, was examined in both groups. For statistical analysis, an independent samples test (Mean ± SD) was used. Results The mRNA levels of LIF (p = 0.045), LIFR (p = 0.040), MUC1 (p = 0.032), VEGF (p = 0.022), EFG (p = 0.035), and FGF2 (p = 0.040) were significantly upregulated in the case group compared with the control group. Conclusion Finally, seminal plasma was observed to be effective in expressing the involved genes in the successful implantation pathway, including LIF, LIFR, MUC1, VEGF, EGF, and FGF2.


Introduction
While several studies have been performed to provide potential molecules, candidates for endometrial receptivity (1)(2)(3)(4)(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 (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.

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.
1. 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.  Table I.

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.

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.

Discussion
The results of this study confirmed that 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.

Conclusion
Finally, it was found that the seminal plasma