Conceived and designed the experiments: NW JDW. Performed the experiments: NW. Analyzed the data: NW LLG. Contributed reagents/materials/analysis tools: BH SCZ JDW. Wrote the paper: NW.
The authors have declared that no competing interests exist.
To explore the molecular basis of the different ultrasonic patterns of the human endometrium, and the molecular marker basis of local injury.
The mRNA and protein expression of FKBP52, progesterone receptor A (PRA), progesterone receptor B (PRB), and HB-EGF were detected in different patterns of the endometrium by real-time RTPCR and immunohistochemistry. There were differences in the mRNA and protein expression of FKBP52, PRB, and HB-EGF in the triple line (Pattern A) and homogeneous (Pattern C) endometrium in the window of implantation. No difference was detected in PRA expression. After local injury, the mRNA expression of HB-EGF significantly increased. In contrast, there was no difference in the mRNA expression of FKBP52, PRB, or PRA. The protein expression of FKBP52, PRB, and HB-EGF increased after local injury. There was no difference in the PRA expression after local injury.
PRB, FKBP52, and HB-EGF may be the molecular basis for the classification of the ultrasonic patterns. HB-EGF may be the molecular basis of local injury. Ultrasonic evaluation on the day of ovulation can be effective in predicting the outcome of implantation.
Ovarian estradiol (E2) and progesterone (P4) are the primary regulators of women's menstrual cycle
P4 plays a critical role in the female reproduction
To play the role of transcriptional activation, PR needs to bind with a chaperone Fkbp52 during the process of implantation. Fkbp52 is an immune-affinity protein containing a tetratricopeptide repeat (TRP) domain that acts as a cochaperone with heat shock protein 90 (HSP90). FKBP52 plays a key role in the implantation of mammals, as confirmed by studies on fkbp null mice
HB-EGF is one of the accepted markers of endometrial receptivity. HB-EGF expression periodically changes in the different stages of a menstrual cycle. The changes are characterized by a low expression during proliferation, a gradual increase after ovulation, and an eventual increase to the peak at the time of implantation
Non-invasive vaginal sonography has been used in clinical diagnosis for many years to evaluate the outcome of implantation
Pattern A endometrium is a typical multi-layered “triple line” that consists of obvious outer and central hyperechogenic lines as well as inner hypoechogenic dark areas. Pattern C endometrium is entirely homogeneous and hyperechogenic. It is characterized by increased reflectivity compared with the myometrium and consequently has a brighter grayscale appearance. Its central hyperechogenic line is not visible. The pregnancy rate is significantly higher in the patients with Pattern A endometrium (30% per cycle) than in those with Pattern C endometrium (9.7% per cycle)
The current study attempts to explore the molecular basis of the different ultrasonic patterns of the human endometrium, and the molecular basis of local injury. The protein and mRNA expression of PRA, PRB, FKBP52, and HB-EGF in Pattern A, Pattern C, and stimulated Pattern C (Pattern SC) endometria are reported.
The twenty women in the current study were divided into Group A and C. Group A had Pattern A endometrium and Group C had Pattern C endometrium. There was no difference between the two groups in their basal characteristics such as age, years of infertility, and menstrual cycle (P>0. 05), as shown in
Group | N | Age (years) | Duration of Infertility (years) | Menstrual Cycle (days) |
A | 11 | 27.73±2.98 | 3.27±0.56 | 29.07±2.22 |
C | 9 | 28.44±2.93 | 3.98±0.37 | 30.22±3.15 |
The serum level of E2 and P4 in Group A, C and SC were all within the normal range. There was no significant difference between Group A and C or Group C and SC (P>0. 05), as shown in
Group or Pattern | N | E2 (pmol/ml) | P(nmol/L) |
A | 11 | 154.29±1.57 | 17.49±2.46 |
C | 9 | 188.82±1.61 | 18.85±1.43 |
SC | 9 | 155.60±1.42 | 13.24±2.34 |
Pattern A was typical triple-layer with central to the outer hyperechoic line between the outer and middle uterine hypoechoic areas or dark areas (
Pattern A endometrium was easy to scrape, elastic, and shiny. Pattern C endometrium was thinner than those of Pattern A, less elastic and shiny, and mostly thin flakes. Pattern SC endometrium improved in thickness, elasticity and shine.
Pattern A endometrium had typical mid-secretory features, rich secretions in gland cavity, jagged glands, stromal edema and had no mitotic figures, (
St = stroma; G = gland; GE = glandular epithelium, 200×magnification.
The mRNA expression of FKBP52, PRB and HB-EGF was significantly higher in Pattern A endometrium than in Pattern C endometrium (P<0.05). There was no difference in the expression of PRA between the endometrium of two patterns (P>0.05), as shown in
The level of PRA, PRB, FKBP52, and HB-EGF mRNA was measured by real-time RT PCR. The values were normalized to GAPDH. A, the mRNA expression was compared between the patients with endometrium of Pattern A and Pattern C by Heteroscedastic T-test. B, the mRNA expression was compared between the same patient with endometrium of Pattern C and Pattern SC by paired T test. *, P<0.05; **, P<0.01.
The mRNA expression of HB-EGF was significantly higher in Pattern C endometrium than in Pattern SC endometrium (P<0.05). There was no difference in the mRNA expression of FKBP52, PRB and PRA between the two patterns (P>0. 05), as shown in
The protein expression of PR and PRB in the endometrium of different patterns was detected by IHC. The cellular distribution of PR was mainly in the nuclear area of stromal cells (
A, B, and C were the staining of the PRA; D, E, and F were the staining of PRB. Positive staining of PRA changed insignificantly in different patterns of endometrium. Positive staining of PRB was weaker in Pattern C endometrium (E) compared with Pattern A endometrium (D), after locally injured, the PRB staining increased (F). St = stroma; G = gland; GE = glandular epithelium, 200×magnification.
H-score was semiquantitatively come from the intensity and distribution of positive staining cells and represented the relative protein level. A, H-score was compared between Pattern A and Pattern C endometrium. B, H-score was compared between Pattern C and Pattern SC endometrium.
In Pattern A endometrium, the protein expression of PRB was mainly distributed in both the glandular epithelium and the stromal cell (
The protein expression of FKBP52 was higher in Pattern A endometrium than in Pattern C endometrium (
A, B, and C were the staining of the FKBP52; D, E, and F were the staining of HB-EGF. Positive staining of FKBP52 and HB-EGF PRB was weaker in Pattern C endometrium (Band E) compared with Pattern A endometrium (A and D), after locally injured, the PRB staining increased (C and F). St = stroma; G = gland; GE = glandular epithelium; LE = luminal epithelium, 200×magnification.
HB-EGF protein was mainly distributed in the plasma of gland epithelial and stromal cells. Consistent with the mRNA expression, HB-EGF was higher distributed in Pattern A endometrium than in Pattern C endometrium (
E2 and P4 are secreted by the ovaries and play important roles throughout the human menstrual cycle. The human endometrium has a strong ability to regenerate and thus plays a key role in the process of implantation
After hematoxylin-eosin (HE) staining, there are differences in the development of endometrium depending on the patterns. The glandular and stromal cells were not fully developed in Pattern C endometrium, with some sub-nuclear vacuoles in glandular epithelia, which is the characteristic of early secretory phase. The nuclei migrated from gland lumen and the edema, as a characteristic marker of the mid-secretory phase, were obvious in the stroma of Pattern A endometrium. In contrast, less edema were observed in Pattern C endometrium. After endometrium was locally injured, the sub-nuclear vacuoles disappeared or decreased in the glands, and the epithelial gland of Pattern SC endometrium increased in secretion. The morphological differences between the two patterns can be caused by the difference in their responsiveness to P4. Local injury can be administered to improve the lag in the development of Pattern C endometrium
PRB, instead of PRA, may be the molecular basis for the classification of the ultrasonic patterns. P4 plays an essential role in female reproduction
In this study, no difference was observed in the expression of PRA between Pattern A and Pattern C endometrium. The same observation was made about the expression of PRA between Pattern C and Pattern SC endometrium. In the mid-secretory phase, the PR expression increased and was mainly located in the nucleus of the stromal cells, while the expression in the glandular epithelium declined sharply and ended up weak. These changes were detected in this study and there was no difference between Pattern A and Patter C or between Pattern C and Pattern SC. This finding verifies the consistency and accuracy of the endometrial samples. The PRB mRNA and protein expression were significantly higher in Pattern A endometrium than in Pattern C endometrium. The PRB differences between Pattern A and Pattern C endometrium were mainly in the stromal cells. These results were consistent with Mulac-Jericevic who suggested that PRB was critical in PR responsiveness to P4
The FKBP52 expression is also related to the classification of the endometrium. To execute the function of P4, PR binds with P4 and other components into a complex including the receptor, Heat Shock Protein (HSP) and cochaperone in the process of implantation
HB-EGF may be the molecular basis of local injury. HB-EGF expression is recognized as a marker of endometrial receptivity. HB-EGF is periodically expressed in the menstrual cycle of human. The expression of HB-EGF is low in the proliferative phase; it increased after ovulation and reaches the highest level at implantation
Various factors may account for the discrepancies between the expression of mRNA and protein levels. Firstly, the trends of mRNA and protein expression were consistent. The increase in protein was more obvious than that in mRNA. The former was statistically significant, while the latter was not. This discrepancy may be caused by the limited size of human samples in the present study. If the sample size is large enough, there is possible that the trend for the expression of RNA and protein levels will be consistent. Secondly, the FKBP52 mRNA expression was detected in the whole endometrial sample, whereas the significant change in FKBP52 protein can be seen only in the grand epithelia. In other words, the change in part of the protein level cannot reflect that of the whole mRNA sample.
In summary, PRB, FKBP52 and HB-EGF are more highly expressed in Pattern A endometrium than in Pattern C endometrium, which partly accounts for the higher pregnancy rate in patients with Patter A endometrium than in those with Pattern C endometrium. This study also suggests that PRB, FKBP52 and HB-EGF may be the molecular basis for the classification of the ultrasonic patterns. Local injury increased the protein expression of PRB, FKBP52 and HB-EGF; however, among the three factors, only HB-EGF underwent a significant increase in its mRNA expression, while no obvious changes were observed of PRB and FKBP52. Therefore, HB-EGF may be the molecular basis of local injury. In conclusion, because the pattern of endometrium on the day of ovulation is closely associated with the expression of PRB, FKBP52 and HB-EGF in the window of implantation, the ultrasonic evaluation of endometrium on the day of ovulation can be effective in predicting the outcome of implantation.
Twenty women sought treatment in the assisted reproduction center of National Research Institute for Family Planning (NRIFP) of China from July to December in 2010. All reported unsuccessful pregnancy as a result of male infertility. The average age of the patients was 28.52 years old. The average duration of infertility was 3.50 years. Institutional review board approval was obtained from the Academic Committee of the National Research Institute for Family Planning on the use of Human Subjects in Medical Research. All the patients provided written informed consent.
Twenty women were divided into two groups Group A and Group C. Group A had Pattern A endometrium as detected by an ultrasound scan. The samples of Pattern A endometrium were collected seven days after ovulation in the spontaneous menstrual cycle. Group C had Pattern C endometrium. The samples of Pattern C endometrium were collected seven days post ovulation when the endometrium was locally injured by curette. In the second spontaneous menstrual cycle, endometrial samples were collected again and the endometrium was named stimulated Pattern C (Pattern SC). Thus, the endometria of Pattern C and Pattern SC were self-controlled. Endometrial biopsies were taken from the anterior wall of the uterine cavity. Each biopsy was divided into two parts. One part of the harvested endometrial pieces was snap frozen in liquid nitrogen and then was stored at −70°C for no longer than six months. The other part was fixed in 4% paraformaldehyde for histochemical analysis. Blood samples were collected on the same day. Serum was separated and stored for the measurement of E2 and P4.
The serum level of E2 and P4 was measured by the enzyme-linked fluorescence assay (ELFA) as described previously
Endometrial samples were collected and stored in liquid nitrogen for RNA isolation. Trizol (Invitrogen) was used to isolate total RNA from human tissue according to manufacturer's protocol. Total RNA was quantified by UV spectrophotometry. One microgram of total RNA was reverse-transcribed by using the GeneAmp RNA PCR system 9700 (Applied Biosystems, Foster City, CA). The final reaction volume was 20 µl with 0.5 ug/µl Oligo (dT) 18. T he reaction conditions were 5 min at 65°C, 1 min at 37°C, 60 min at 50°C, and 15 min at 70°C. Real-time PCR was performed by using the following primer sequences:
PRA: F-
PRB: F-
FKBP52: F-
After 3 min of incubation at 95°C, 40 cycles were performed as follows: denaturation at 94°C for 20 s, annealing at 59°C for 20 s and extension at 72°C for 30 s. The results are normalized to the amount of GAPDH and expressed as abundance by the ΔCt method
The endometrial samples were processed by conventional preparation for histology and cut into 5 µm sections. HE staining was used to histological evaluate of the endometrial biopsies according to the criteria of Noyes et al
For IHC, mouse anti-human PR (1A6) (NCL-PGR, Leica, USA) antibody was diluted 1∶ 200 in antibody dilution, and the rabbit anti human PRB (MS-192-P1, Thermo Scientific, UK) was diluted at 1∶250. The FKBP52 antibody (ab84536, Abcam, UK) was diluted at 1∶300, and the HB-EGF (AF-259-NA, R&D) was diluted at 1∶200. Sections were deparaffinized, rehydrated and washed in 0.1 M PBS (pH 7.4). The sections were then immersed in 3% hydrogen peroxide to block endogenous peroxidase and incubated for 5 to 10 minutes. Slides were incubated in antibodies against human PR, PRB, HB-EGF and FKBP52 at 4°C overnight. Signal was detected by adding biotinylated secondary antibodies and streptavidin-peroxidase, and stained using 3, 3′-diaminobenzidine plus peroxide solution.
The IHC staining of the four detected factors (PR, PRB, HB-EGF and FKBP52) was scored semiquantitatively by using the quick score method as described
Average mean analysis was determinate by student's T test using the computer program SPSS 11.5. A value of P<0.05 was considered statistically significant.