Embryo implantation: Shedding light on the roles of ovarian hormones, cytokines and growth factors in the implantation process

Implantation is a crucial step in mammalian reproduction, as it is a gateway to further embryonic  development and successful pregnancy. Successful implantation requires coordinated interactions between the blastocyst and uterus. Uterine receptivity for embryo implantation is regulated by the ovarian hormones estrogen and progesterone. Some cytokines and growth factors play important roles in embryo implantation under the influence of ovarian hormones. Such molecules are involved in embryo-maternal interactions during the implantation process. This review describes the implantation process and highlights the potential roles of some cytokines and growth factors (such as leukemia inhibitory factor, interleukin-1, interleukin-6, interleukin-11, colony stimulating factor-1, epidermal growth factor family and insulin-like growth factor system) in the implantation process. Knowledge of the function of these molecules during implantation may help to address the reason of implantation failure and infertility.Key words: Implantation, estrogen, progesterone, cytokines, growth factors

Morphological and molecular changes in embryo-maternal interactions in a rat model at optimal and elevated environmental temperatures

Early embryonic development, successful implantation, optimal fetal and placental development, and maintenance of a pregnancy are critically dependent on intact embryo-maternal interactions. Today, assisted reproductive technology (ART) is widely used to overcome some causes of infertility; however, the success rate remains low as a result of failures in communication between the transferred embryo and the endometrium. Failure in embryo-maternal communication is responsible for reproductive wastage, resulting in enormous economic loss. The mechanism that regulates embryo-maternal interaction has remained elusive and is not well understood. A better understanding of early embryo-maternal communication and the identification of the external factors that could interfere with embryo-maternal crosstalk will improve reproductive success in both humans and animals. The present study focuses on the maternal signals, which are responsible for two-thirds of pregnancy losses. Thus, the main objectives of this study were to define the morphological and molecular changes that occur in the maternal tissue in response to the presence of the embryo and to determine the effects of elevated ambient temperatures on the morphological and molecular responses of maternal tissue to embryonic signals. In the present study, implantation time was determined using blue dye injection (1% Chicago Sky Blue 6B). A radioimmunoassay (RIA) was used to estimate the plasma estradiol and progesterone levels during peri-implantation. Morphological changes in the rat oviduct and uterus during early pregnancy were examined under light and electron microscopes. Localization of transforming growth factor β1 (TGFβ1), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors TGFβR1, VEGFR2 (Flk-1), FGFR1 (Flg) in the oviduct and uterus was determined by immunohistochemistry. TGF β1, VEGF, and bFGF gene expression levels in the rat uterus were examined by real-time quantitative RT-PCR. The effects of elevated ambient temperatures on implantation time, number of implantation sites, plasma estradiol and progesterone concentrations, morphological changes, and the expression levels of TGFβ1, bFGF, and VEGF in the uterus during peri-implantation were also examined. Further, the effects of exposure to elevated temperatures at different stages of pregnancy on gestation length, litter size, neonatal deaths, sex ratio, birth weight, and offspring growth were also determined. In the present study, implantation was initiated on day 5 and established by day 7 of pregnancy in the rats kept under optimal temperatures (23±1°C), while the rats exposed to elevated temperatures (33±2°C) exhibited delayed implantation and a reduced number of implantation sites. Moreover, the exposure to elevated temperatures resulted in changes in the patterns of plasma progesterone and estradiol levels during peri-implantation. Exposure to elevated ambient temperatures during early pregnancy resulted in prolonged gestation, reduced litter size, increased neonatal death, and a sex ratio biased toward males. Moreover, elevated temperatures adversely affected the birth weight and growth of offspring. Exposure to elevated ambient temperatures during the pre- and peri-implantation periods had stronger adverse effects on reproductive outcomes and offspring growth than post-implantation exposure. Rats maintained under optimal conditions exhibited many morphological changes in the oviduct and uterus during early pregnancy. In the oviduct, the secretory cells were predominant during the first 4 days of pregnancy. On days 5 through 8 of pregnancy, however, the ciliated cells were predominant. In the uterus, extensive infiltration of leukocytes was observed in the endometrium during pre-implantation, whereas during implantation, the number of leukocytes decreased. Additionally, considerable changes occurred in the apical plasma membrane of the uterine epithelial cells. Such changes included changes in the length and density of the microvilli, flattening of the uterine epithelial cells, loss of the small secretory droplets seen near the epithelial cells, and the appearance of cytoplasmic protrusions (pinopods). The uterine luminal epithelial cells gradually lost their microvilli and became very flat. Pinopods were observed at the apical surface as early as day 2 of pregnancy. The incidence of pinopods increased gradually up to day 5 of pregnancy, when the pinopods became abundant and appeared in doughnut shaped.In rats exposed to elevated ambient temperatures, there was a reduced incidence of pinopods on day 5 compared to the rats maintained under optimal temperatures. On day 6 of pregnancy, the rats exposed to elevated temperatures did not show flattening of the apical membrane. Immunohistochemistry showed that TGFβ1, TGFβR1, VEGF, VEGFR2 (Flk-1), bFGF, and FGFR1 (Flg) were expressed in both the oviduct and uterus throughout the first 8 days of pregnancy; however, the distribution and intensity of immunostaining for each protein varied depending on the day of pregnancy. In the oviduct, high immunoreactivities of these growth factors and their receptors were observed while the embryo was present in the oviduct. In the uterus, TGFβ1, TGFβR1, VEGF, Flk-1, bFGF, and Flg were detected in the endometrium, i.e., in the epithelial and stromal cells. Both immunohistochemical analysis and real time RT-qPCR revealed that TGFβ1, VEGF, and bFGF were found in the uterus at relatively lower levels during preimplantation but that their expression levels increased dramatically during periimplantation. Expression of TGFβ1 increased significantly on day 5 (8.72 ± 1.20 - fold increase versus day 0, P < 0.001) and day 5.5 (13.22 ± 1.80 - fold increase versus day 0, P < 0.001). A dramatic increase in TGFβ1 expression was detected on day 6 of pregnancy (46.38 ± 8.57 - fold increase versus day 0, P < 0.0001). Elevated expression of bFGF was observed immediately before implantation on day 5 (13.04 ± 1.79 - fold increase versus day 0, P < 0.0001), whereas VEGF showed elevated expression on day 5.5 (52.40 ± 6.50 - fold increase versus day 0, P < 0.0001).The TGFβ1 mRNA levels in the uteri of rats exposed to elevated temperatures on days 5, 5.5, and 6 of pregnancy were significantly (P < 0.01) lower than the expression levels in the uteri of rats kept under optimal temperatures at the same stages of pregnancy. VEGF and bFGF expression in the uteri of rats exposed to elevated temperatures on days 5 and 5.5 were significantly (P < 0.01) lower than the expression levels in uteri of rats maintained under optimal temperatures at the same stages of pregnancy, while high expression levels were observed on day 6 of pregnancy. In conclusion, implantation in rats was initiated on day 5 and established by day 7 of pregnancy. Elevated ambient temperatures can delay implantation and reduce the number of implantation sites. Elevated temperatures disturb the plasma estradiol and progesterone levels during peri-implantation. Elevated temperatures can also lead to prolonged gestation time, decreased litter size, neonatal death, and a sex ratio biased toward males. Additionally, exposure to elevated temperatures during early pregnancy has adverse effects on offspring growth. The exposure of pregnant rats to elevated temperatures during the pre- and peri-implantation stages has stronger adverse effects on reproductive performance than post-implantation exposure. Morphological alterations in the apical plasma membrane indicate endometrial receptivity. The expression patterns of TGFβ1, VEGF and bFGF and their receptors in the oviduct indicate that these growth factors contribute to early embryonic development. High expression levels of TGFβ1, VEGF, and bFGF during periimplantation suggest that these growth factors play roles in implantation in rats. Exposure of pregnant rats to elevated temperatures affects the responses of uterine tissue (i.e., both morphological and molecular responses) to embryonic signals,leading to delay or failure of implantation. Overall, this study reveals that morphological alterations and growth factors (TGFβ1, VEGF, and bFGF) are involved in embryo-maternal interactions and that elevated environmental temperatures interfere with the embryo-maternal crosstalk during peri-implantation, leading to implantation delay/failure. These findings can help in the diagnosis and treatment of non-receptive endometria and abnormal embryo-maternal interactions

Reproductive characteristic of the female laboratory rat

The laboratory rat is widely used in research studies as a model of mammalian health and disease. This review provides the basic facts about female rat reproduction and highlights the reproductive characteristics which make the rat a preferred animal model for research on reproduction. Rats have short estrous cycle, lasting four to five days. The estrous cycle consists of four stages known as proestrus, estrus, metestrus and diestrus. Phases of the estrous cycle can be detected by observing behavioral changes or examining vaginal cytology. Detection of sperm in vaginal smear is an excellent predictor of pregnancy in rats. Implantation is initiated on day 5 and completed by day 7 of pregnancy. Gestation takes 21 to 23 days from copulation to parturition. Short estrous cycle and gestation period make the rat an ideal animal model for research on reproduction

Effects of Elevated Ambient Temperature on Reproductive Outcomes and Offspring Growth Depend on Exposure Time

Reproductive performance has been shown to be greatly affected by changes in environmental factors, such as temperature. However, it is also crucial to identify the particular stage of pregnancy that is most adversely affected by elevated ambient temperature. The aims of this study were to determine the effect on reproductive outcomes of exposure to elevated ambient temperature during different stages of pregnancy and to determine the effect of prenatal heat stress on offspring growth. Sixty pregnant rats were used in this study. The rats were divided equally into four groups as group 1 (control), group 2 (exposed to elevated temperature following implantation), group 3 (exposed to elevated temperature during pre- and periimplantation), and group 4 (exposed to elevated temperature during pre- and periimplantation and following implantation). Groups 3 and 4 had prolonged gestation periods, reduced litter sizes, and male-biased sex ratios. Moreover, the growth patterns of group 3 and 4 pups were adversely affected by prenatal exposure to elevated temperature. The differences between group 1 and group 3 and between group 1 and group 4 were highly significant. However, no significant differences were observed between groups 1 and 2 in the gestation length, sex ratios, and growth patterns. Thus, it can be concluded that exposure to elevated ambient temperature during pre- and periimplantation has stronger adverse effects on reproductive outcomes and offspring growth than postimplantation exposure

Effects of elevated ambient temperature on embryo implantation in rats

Implantation is a crucial step in mammalian reproduction as it is a gateway to further embryonic development and successful pregnancy. Changes in the environmental factors, such as temperature have adverse effects on reproduction. However, the impact of elevated temperature on the implantation process is not well defined. The objective of this study was to investigate the possible effect of elevated ambient temperature on implantation time and rate. The results revealed that exposure to elevated ambient temperature leads to a delayed implantation and reduced number of implantation sites in Sprague Dawley rats. Moreover, the exposure to elevated temperature resulted in change in the progesterone and estradiol patterns during the implantation time. These findings indicate that elevated temperature disturbs the implantation process