DOI 10.1007/s10517-015-2861-5 Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015
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GENETICS Profiles of mRNA Expression for Genes Involved in Implantation, Early and Middle Phases of Secretion Stage in Human Endometrium M. A. Maslova, V. Yu. Smol’nikova, A. M. Savilova, O. V. Burmenskaya, A. A. Bystritskii, V. K. Tabolova, I. E. Korneeva, T. A. Demura, and A. E. Donnikov Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 12, pp. 748-751, December, 2014 Original article submitted October 21, 2014 The expression of mRNA of 36 genes involved in implantation was studied by reverse transcription and real-time PCR. Significant differences in mRNA expression during the early and middle stages of the secretion phase were detected for genes mmp7, vegf, il2m, il1β, il8, il18, tnfα, il10, tgfβ, igfbp2, etc. Key Words: endometrium; gene expression; mRNA; real time polymerase chain reaction The interests of researchers investigating the problems of basic reproductive medicine are focused on the endometrium, specifically, on the spectrum of potential receptivity factors and structural disorders (hyperplasia, hypoplasia, formation of intrauterine synechiae). Genes encoding factors of the cell adhesion family, IL-6 cytokine family, vascular endothelial growth factor (VEGF), and other genes are assumed to be involved in this process. However, the role of the potential biomolecular markers in the implantation process is little studied for ethic and technological reasons, and the majority of studies in this field are performed on rodents and other animals. The key role of endometrial morphology and function in effective implantation of the embryo necessitates comprehensive evaluation of the endometrium status prior to in vitro fertilization (IVF) program in order to choose the pathogenetic therapy and the strategy of preparation to pregnancy. In the search for optimal criteria of the endometrium readiness to implantation highly informative V. I. Kulakov Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of the Russian Federation, Moscow, Russia. Address for correspondence:
[email protected]. A. M. Savilova
molecular diagnostic methods in combination to histological studies are used. The attention of fertility specialists is traditionally focused on the so-called implantation window, the period when the endometrium is ready to “accept” the blastocyst. The implantation window falls on days 6-8 after ovulation (or ovarian puncture in IVF program). From histological viewpoint, the middle stage of the secretion phase of the menstrual cycle corresponds to this period. Even minor deviation from this stage is essential for endometrial readiness to implantation; however, precise histological verification is often neglected during the implantation window, which can distort significantly the results of studies. We studied the profiles of mRNA expression for genes involved in implantation in the endometrium during the early and middle phases of secretion.
MATERIALS AND METHODS A cohort prospective study of endometrial specimens was carried out. The specimens were collected in 117 women aged 24-42 years (median 33.1±3.9 years) with tuboperitoneal sterility treated by the IVF method. All
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patients signed informed consent to participation in the study. The inclusion criteria were age 18-42 years, regular cycle, intact ovarian reserve, and the absence of endometrial disease. The exclusion criteria were infertility of immunological origin, uterine myoma of any size, acute inflammatory diseases of the pelvic organs, developmental abnormalities of the internal genitals, and somatic and mental diseases. Aspiration pipelle biopsy of the endometrium by the Pipelle de Cornier (Laboratorie C.C.D.) was carried out in all patients in the middle of the lutein phase and morphological and immunohistochemical (IHC) studies of the samples were carried out. Biopsy specimens were fixed in 10% neutral formalin (24 h), processed by standard methods, and embedded in paraffin. The expression of estrogen α and progesterone receptors, leukemiainhibiting factor (LIF) in the glands and stroma were evaluated by IHC reactions in paraffin sections (4-5 μ) by the routine protocols (DAKO protocols). For molecular genetic studies, the biopsy specimen was plunged in tubes with a medium for RNA stabilization (Proba-NK system; DNA Technology). mRNA expression was analyzed for 36 genes involved in the regulation of implantation, among them genes of cytokines, growth factors, metalloproteinases, surface markers, oncomarkers, estrogen and progesterone receptors, and homeobox-containing genes (Table 1). The level of mRNA expression was evaluated by the real time reverse transcription PCR using commercial test systems (DT-96 detecting amplifier; DNA-Technology). The gene expression levels were standardized by comparing the indicator cycles (∆∆Cq) by hprt1, tbp, b2m, and gusb reference genes [1]. The median (Me) was selected as the measure of the central trend of quantitative signs, the upper (L) and lower (N) quartiles were used for interval evaluation. The results were presented as the Me (25-75%). The significance of differences between the groups was evaluated by Mann–Whitney U test for unrelated groups.
RESULTS Morphological studies of endometrium biopsy specimens showed the early stage of secretion phase in 55 (47%) patients (group 1) and middle stage in 62 (53%) patients (group 2). Studies of gene mRNA expression in the endometrium showed significant differences in mRNA levels depending on the secretion phase (Table 1). The level of mRNA expression differed significantly at different secretion stages for the majority of genes. Generally, the expression was significantly (23-fold) higher during the middle in comparison with the early stage (vegf, il1β il8, il15, tnfα, cd56, il6, il10, osm, mmp7, cd45, cd68, et al.). The differences were much more significant for some gene mRNA: the lev-
els of igfbp1 and lif mRNA increased 50- and 12-fold, respectively, during the middle secretory phase. The expression of some gene mRNA (lifr, hox7) somewhat decreased. These changes in mRNA expression revealed by us could be attributed to not only variation of the levels of gene mRNA in endometrial cells, but also variation in proportion of cells of different types infiltrating the endometrium during different phases. For instance, il6, tgfβ, il1β, il8, and vegf genes could be expressed in endometrial stromal cells and in lymphocytes present in the vessels and infiltrating the stroma. Protein products of cd56 (NCAM) and cd68 genes are present on immune system cells (NK and monocytes/macrophages, respectively). Hence, the method used in the study does not answer the question whether the changes in the expression of gene mRNA was due to changes in their levels in endometrial cells (stromal, epithelial, or glandular) or changed proportion between different cell types infiltrating the endometrium during different phases of the menstrual cycle. However, the data suggest that secretion phase should be taken into account when determining the implantation window reflecting important hormone-mediated changes. The differences in the expression of gene mRNA and protein products in the endometrium during different phases of the cycle were studied not once. In 1995, significant variations in the levels of protein molecules expression during different phases have been demonstrated by 2D-electrophoresis [5], but the proteins were not identified. A later study found that the levels of il6 gene mRNA and of its protein product changed significantly over the cycle: the expression was low during the proliferative phase and increased 5-10-fold during the middle and late secretory phases [6]. This was in line with our findings: the level of il6 gene expression was 2-fold higher during the middle than during the early phase of secretion. VEGF, the key angiogenic factor in the endometrium, promotes the increase in vascular permeability, suppresses apoptosis, stimulates NO and prostacyclin release by endotheliocytes in spiral arteries. This factor is expressed mainly in the endometrial glandular epitheliocytes and diffusely in the stroma, the peak of its expression observed during the middle of the secretory phase, while the decrease in VEGF-A expression during the middle phase of secretion was detected in patients with repeated IVF failure without organic abnormalities of the endometrium [4]. Hence, disorders in the angiogenesis process associated with low expression of vegf gene can lead to disorders in cyclic transformation of the endometrium, implantation failure, and miscarriages. Blastocyst implantation (the process similar to the inflammatory reaction) is associated with enhanced
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TABLE 1. Gene mRNA Expression in Endometrium during Different Phases of Secretion Stage Gene
Group 1 (N=55)
Group 2 (N=62)
р
hox-7
0.32 (0.23-0.53)
0.18 (0.11-0.37)
0.00007
vegfa
0.17 (0.09-0.24)
0.32 (0.19-0.47)
1.77×10–7
vegfa189
0.12 (0.05-0.18)
0.19 (0.12-0.33)
0.002
0.006 (0.004-0.123)
0.013 (0.008-0.298)
0.00002
lif
0.00026 (0.00003-0.00149)
0.00324 (0.00069-0.00866)
8.36×10–7
lifr
0.09 (0.06-0.14)
0.07 (0.05-0.01)
0.027
il8
0.004 (0.001-0.008)
0.007 (0.003-0.016)
0.003
il15
0.08 (0.04-0.12)
0.15 (0.09-0.28)
6.79×10–7
il18
0.014 (0.009-0.022)
0.018 (0.013-0.024)
0.014
tnfα
0.002 (0.001-0.004)
0.005 (0.002-0.007)
0.00004
cd56
0.0018 (0.0007-0.0047)
0.0028 (0.0011-0.0049)
0.028
cox-2
0.019 (0.012-0.031)
0.008 (0.004-0.022)
0.001
igfbp1
0.000007 (0.000001-0.000105)
0.000365 (0.000026-0.002377)
2.83×10–7
0.07 (0.04-0.19)
0.24 (0.13-0.37)
0.005
il6
0.00015 (0.00002-0.00037)
0.00025 (0.00013-0.00079)
0.014
il10
0.00013 (0.00005-0.00032)
0.00023 (0.00015-0.00042)
0.019
osm
0.0007 (0.0003-0.0018)
0.0016 (0.0012-0.0030)
0.002
mmp7
0.05 (0.02-0.10)
0.12 (0.06-0.32)
0.003
cd45
0.01 (0.01-0.02)
0.02 (0.01-0.03)
0.016
il1β
cd68
pten
1.06 (0.08 -1.30)
1.29 (1.00-1.70)
0.008
igfbp2
0.102 (0.055-0.118)
0.148 (0.092-0.198)
0.001
hoxa10
0.28 (0.21-0.40)
0.31 (0.20-0.41)
nd
hoxa11
0.22 (0.10-0.32)
0.23 (0.14-0.37)
nd
0.00008 (0.00004-0.00011)
0.00009 (0.00006-0.00015)
nd
0.003 (0.001-0.009)
0.004 (0.001-0.010)
nd
0.15(0.08-0.22)
0.14 (0.11-0.21)
nd
mmp2
0.152 (0.007-0.026)
0.015 (0.010-0.021)
nd
mmp9
0.0004 (0.0001-0.0013)
0.0008 (0.0003-0.0021)
nd
il2 mmp11 tgfβ
igf2
0.0012 (0.0001-0.0062)
0.0012 (0.0005-0.0167)
nd
bcl2
0.003 (0.001-0.007)
0.004 (0.002-0.012)
nd
bax
0.004 (0.002-0.009)
0.006 (0.004-0.009)
nd
mmp8
0.00004 (0.00001-0.00006)
0.00005 (0.00002-0.00009)
nd
igfbp4
0.09 (0.03-0.29)
0.14 (0.07-0.43)
nd
0.0004 (0.0003-0.0006)
0.0004 (0.0003-0.0006)
nd
il12a
Note. nd: differences are not significant.
production of proinflammatory Th1 cytokines in the endometrium; one of them, IL-15, induces activation and proliferation NK cell in the endometrium essential for implantation (regulation of the maternal immune response to trophoblast invasion, remodeling of spiral arteries, and differentiation of endometrial stromal
cells). In addition, NK cells are the sources of various cytokines and growth factors. The level of il15 mRNA in the endometrium increases during the lutein phase and reaches the peak during the implantation window [2]. The increase in IL-15 level correlates with increase in the count of NK cells in the endometrium,
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their excess leading to infertility, implantation failure, and habitual miscarriages [3]. Our findings are essential for the creation of molecular genetic tests for pregavid evaluation of the endometrial status assessing endometrial receptivity and for isolation of cell cultures from the endometrium for research and practical purposes. The material should be collected with due consideration for the phases of the menstrual cycle in order to obtain reproducible correct results and homogenous endometrial cell cultures. The study was supported by the Russian Scientific Foundation (grant No. 14-25-00179).
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