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NLF2 gene expression in the endometrium of patients with implantation failure after IVF treatment
Gene 508 (2012) 140–143
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
NLF2 gene expression in the endometrium of patients with implantation failure after IVF treatment Esengul Turkyilmaz a, Haldun Guner a, Mehmet Erdem a, Ahmet Erdem a, Aydan Asyali Biri a, Ece Konac b,⁎, Ebru Alp b, Hacer Ilke Onen b, Sevda Menevse b a b
Department of Obstetrics and Gynecology, Faculty of Medicine, Gazi University, 06500 Besevler, Ankara, Turkey Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, 06500 Besevler, Ankara, Turkey
a r t i c l e
i n f o
Article history: Accepted 18 July 2012 Available online 3 August 2012 Keywords: Repeated implantation failure In vitro fertilization Gene expression NLF2 MFAP2 MFAP5
a b s t r a c t The aim of this study was to analyze the expression of microfibril-associated protein 2 (MFAP2), microfibril-associated protein 5 (MFAP5) and nuclear localized factor 2 (NLF2) genes in patients with repeated IVF failure and compare with fertile population. Total RNA was isolated from 38 patients (repeated implantation failure, group 1, n = 22; fertile patients, group 2, n = 16). mRNA expression levels were measured quantitatively using real-time polymerase chain reaction. Our results showed that mRNA expression of NLF2 significantly decreased in the infertility group as compared to control group (P = 0.023). In addition a marked decrease was observed in the expression of MFAP2 in women with repeated implantation failure. In conclusion, NLF2 gene expression levels and differences in MFAP2 and MFAP5 gene expressions (albeit being insignificant) between infertile group and control group draw attention to a genetic basis under implantation failure. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Endometrium becomes receptive to blastocyst implantation for a limited period called “the implantation window”, 6–8 days after ovulation and remains receptive for 4 days (Wilcox et al., 1999). This limitation in time leads to low implantation rates even in natural cycles. Despite many advances in assisted reproductive technologies (ART), low implantation rate is still the most important factor negatively affecting success rates. Successful implantation depends on synchronization between the developmental stages of an embryo itself and the complex molecular and cellular events induced by paracrine and autocrine regulators in endometrium within the implantation window. Endometrial biopsy and dating of the endometrium according to the classical criteria of Noyes were once used widely to ascertain that the ovulation has Abbreviations: ANGPTL, angiopoietin-like protein 1; APCR, activated protein C resistance; ART, assisted reproductive technology; DKK1, Dickkopf-related protein 1; ECM, extracellular matrix; EG-VEGF, endocrine gland-derived vascular endothelial growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HB-EGF, heparin-binding EGF-like growth factor; ICSI, intra cytoplasmic sperm injection; IL-1, interleukin-1; IL-11, interleukin-11; IVF, in vitro fertilization; LEF1, lymphoid enhancer-binding factor 1; LH, luteinizing hormone; LIF, leukemia inhibitory factor; MAGP2, microfibril-associated glycoprotein 2; MFAP2, microfibrillar-associated protein 2; MFAP5, microfibrillarassociated protein 5; NLF2, nuclear localized factor 2; PCR, polymerase chain reaction; QRT-PCR, quantitative real time PCR; SFRP1, secreted frizzled-related protein 1; TSH, thyroid-stimulating hormone; UPL, universal probe library. ⁎ Corresponding author. Tel.: +90 312 202 46 34; fax: +90 312 212 46 47. E-mail address: [email protected] (E. Konac). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.07.031
occurred as well as to ensure that endometrium is in proper histological synchronization (Noyes et al., 1975). However, it was found that ‘out of phase’ endometrium was also frequently encountered in endometrial biopsy of fertile as well as infertile women (Coutifaris et al., 2004). Advances in molecular biology provided additional information regarding the different expression patterns of many genes through out implantation window. Many molecules including integrins, mucins, growth factors (HB-EGF), cytokines (LIF, leptin, IL-1, IL-11), and lipids have recently been identified as important markers of implantation process. Expression profiles of many genes and their products were used as markers of endometrial receptivity and implantation with the help of microarray technology in endometrial biopsy specimens. In a recent study, microfibril-associated protein 5 (MFAP5) and nuclear localized factor 2 (NLF2) were suggested as new candidate genes for markers of human implantation in natural cycles (Haouzi et al., 2009a). MFAP5 gene, also called microfibril-associated glycoproteins 2 (MAGP2), encodes a microfibril-associated glycoprotein, which is a component of microfibrils, an important structural component of elastic tissues, such as vasculature. This protein is located in a position that would allow it to potentially modulate cell‐matrix interactions and participate in cell signaling pathways by interacting with both extracellular matrix (ECM), such as collagen and cell-associated proteins, such as integrins, (Lemaire et al., 2005; Miyamoto et al., 2006). It has also been suggested that MFAP5 has a role in “Notch signaling activation”; a pathway involved in vascularization during embryogenesis, development and normal homeostasis (Haouzi et al., 2009a). MAGP-2 promotes angiogenic cell sprouting by antagonizing
E. Turkyilmaz et al. / Gene 508 (2012) 140–143
“Notch signaling” pathways in endothelial cells in vitro (Albig et al., 2008). MFAP2 gene, also called MAGP1, is a regulator gene of bone remodeling and its absence is associated with osteopenia. NLF2 was identified as a nuclear factor and a member of a regulatory genes family that plays a role in endothelial cell inflammation. It has been suggested that NLF2 is probably a part of the signaling pathway causing changes in cell architecture and adhesion in endothelial cell inflammation (Warton et al., 2004). Recently it has been shown that the NLF2 gene was strongly expressed in the endometrium of luteal day LH + 7 samples, emphasizing its role in endometrium remodeling during implantation window (Haouzi et al., 2009a). Implantation remains a rate-limiting step for the success of the in vitro fertilization (IVF) treatments. No data exist, in our knowledge, regarding the role of MFAP5, MFAP2 and NLF2 gene expressions in patients with implantation failure after repeated IVF attempts. We aimed to compare the expression of these genes in patients with repeated IVF failure with normal fertile population. 2. Materials and methods 2.1. Study design, patient selection and tissue collection All patients in the study group had primary infertility and recurrent implantation failures after IVF. Implantation failure was defined as failure to conceive after one or more IVF attempts in which at least one good quality embryo was transferred. The Ethics Committee of Gazi University Faculty of Medicine approved study and informed consents were obtained from all subjects. All patients in the study group had patent fallopian tubes confirmed by hysterosalpingography or laparoscopy. Patients with unilateral tubal occlusion were included in the study group. Patients with factors or lesions that interfere negatively with implantation, such as severe male infertility and intracavitary lesions like polyps, fibroids, intrauterine adhesions and patients with uterine anomalies were excluded from the study group. Patients with endometriosis were excluded from the study. Patients with anovulation were also excluded since only endometrial biopsies in ovulatory patients can reveal secretory endometrial pattern. Control group was composed of fertile, ovulatory women who had at least one term live birth without any previous clinical pregnancy loss. Both groups were evaluated for thyroid functions and thrombophilic factors. Serum TSH levels were measured in all patients before recruiting them for the study. All women were tested for the presence of Factor V Leiden, prothrombin (G20210A) and methylenetetrahydrofolate reductase (C677T) mutations as well as for deficiencies in proteins S, C and antithrombin III and for resistance to activated protein C. Endometrial biopsy samples were obtained from the anterior wall of the uterine cavity using a “Pipelle catheter”. Samples were obtained during midsecretory phase‐day 7 in patients after endogenous luteinizing hormone (day LH + 7) peak or day 6 in patients after documentation of ovulation by ultrasonography. Serum progesterone levels were measured in all patients on the day of biopsy and patients with normal midluteal progesterone levels were included in the study. Any serum progesterone level greater than 6 ng/ml provides objective evidence that ovulation has occurred (Wathen et
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al., 1984). Histological evaluation of endometrial samples were made using the criteria described by Noyes et al. and only samples with normal maturation in relation to the corresponding cycle day were used for evaluation of gene expressions (Noyes et al., 1975). 2.2. Total RNA extraction and reverse transcription PCR (RT-PCR) Total RNA was isolated from endometrial tissue (approximately 50–100 mg) using the Trizol reagent (peqGOLD TriFast™, peqlab, Erlangen, Germany) as previously described by Konac et al. (2009). To avoid DNA contamination, we applied some modifications to manufacturer's instructions. RNA-containing pellet was treated with approximately 1–5 U RNase-free DNase per μg RNA and incubated at 37 °C for 30 min before being washed with 75% ethanol. The yield and quality of the RNA of each sample was determined by measuring the absorbance at 260 and 280 nm using the “NanoDrop spectrophotometer” (NanoDrop ND-1000, Montchanin, DE, USA). Total RNA (1 μg) was reverse-transcribed in a 20 μl reaction mixture using random hexamers and Transcriptor First Strand cDNA synthesis kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. 2.3. Quantitative real-time PCR (QRT-PCR) analysis MFAP2, MFAP5 and NLF2 mRNA expression levels were measured using the real-time PCR method. Probes and primer sets for each gene were designed at the ProbeFinder. Assay Design Center website (https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp? id=uplst 030000). PCR primers (exon–exon junction to allow discrimination between cDNA and gDNA) and Universal Probe Library (UPL) numbers for this study are provided in Table 1. The 10 μl reaction mixture, prepared in borosilicate glass capillaries, contained 1 × LightCycler TaqMan Master reaction mixture (Roche Diagnostics, GmbH, Mannheim, Germany), 2.5 pmol of each primer, 1 pmol of UPL probe, 4 mM MgCl2 and 1 μl cDNA. The real-time PCR assay included a no-template control. All PCR reactions were performed in the LightCycler 1.5 instrument (Roche Diagnostics, GmbH, Mannheim, Germany) using the following program conditions: 95 °C for 10 min followed by 45 cycles at 95 °C for 10 s , 60 °C for 20 s and finally a cooling step to 40 °C. We analyzed the expression of GAPDH housekeeping gene to normalize the results obtained by QRT-PCR. Each sample was tested in triplicate. PCR efficiency for each gene was tested by serial dilutions and amplification efficiencies of target genes and GAPDH were approximately equal. 2.4. Statistical analysis Mann–Whitney U test was performed using SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA) to compare both groups with regard to age, sampling day, endometrial date according to histological findings, serum progesterone and thyroid hormone levels and levels of acquired thrombophilia factors. Fisher's exact test and Chi-square test were used to compare both groups with regard to gene mutations resulting in thrombophilia. A P-valueb 0.05 was considered significant. All values were expressed as mean ± standard deviation (SD). Statistical
Table 1 The gene-specific primer sequences and probe numbers. Gene
Forward primer
Reverse primer
UPL probe no.
GAPDH MFAP2 MFAP5 NLF2
5′-AGCCACATCGCTCAGACAC-3′ 5′-TGCAAACAGTGTCTCAACGA-3′ 5′‐GGAACACGAAGCTATGAAAGATG-3′ 5′-GCTTGCAACCAGATCCAGAG-3′
5′-GCCCAATACGACCAAATCC-3′ 5′-CTTGTCCCGACAGAGGTCAG-3′ 5′-GGGTCTCTGCAAATCCACAT-3′ 5′-GGCCGAGGAACAGAGTTTC-3′
60 75 69 75
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significance levels of differences in mRNA expressions were analyzed by relative expression software tool (REST 2008 version 2.07). 3. Results All patients in the study group had primary infertility. Mean infertility duration was 8.8 ± 5.1 years (minimum 3; maximum 20 years). All patients had repeated IVF failure during a period of 1 to 5 years. 22 patients had unexplained infertility. Mean sperm count was 31.7 × 10 6 /mL. (± 27.3 × 10 6) (range 1–90 × 10 6) and percentage motility was 43.8 ± 17.4 (range 24%–43%). There was no difference among patients and controls in terms of age, sampling days, serum progesterone levels, thyroid function tests and thrombophilia parameters except for activated protein C resistance (P > 0.05). Detailed characteristics of the sample study population and the P-values are reported in Table 2. “Activated protein C” resistance (APCR) level was detected higher than normal level in 10 patients from the infertile group and 4 patients in the fertile group and the difference was statistically significant. Protein C, protein S and antithrombin III (AT III) values were not different between two groups. mRNA expression of MFAP2 was decreased in implantation failure group as compared to control group (1.34 fold change; P = 0.362). In contrast there was a 1.37-fold increase in the expression level of MFAP5 in the infertile patients with implantation failure as compared to control group (P = 0.138). The expression level of NLF2 decreased 1.84 folds in the implantation failure group as compared to the control group (P = 0.023). Comparison of MFAP2, MFAP5 and NLF2 gene expressions was demonstrated in Fig. 1. 4. Discussion In this study, expression profiles of MFAP5, MFAP2 and NLF2 genes were investigated in endometrial samples during the implantation window in a group of patients with implantation failure defined by multiple unsuccessful IVF attempts and the findings were compared with those of fertile controls. Our results showed that mRNA expression of NLF2 significantly decreased in the infertility group as compared to control group. The gene expression profiles of MFAP2 and MFAP5 were not different between infertile and fertile groups. Implantation is a process which involves attachment of the blastocyst to endometrium, creating an interaction between growing embryo and maternal circulation (Aplin 2000; Denker 1993). Many genes and molecules play significant roles in the implantation process. MFAP2, MFAP5 and NLF2 were recently recognized; thus their roles in the implantation process are yet to be determined. Table 2 Mean clinicopathologic characteristics of patients and controls.
Age Sampling day Histologic dating to Noyes criteria Serum progesterone (ng/ml) Serum TSH (mIU/L) Serum free T3 (pg/ml) Serum free T4 (ng/dl) AT III (%) Protein C (%) Protein S (%) APCR (%) No of previous in vitro fertilization cycles Sperm concentration (×106)
Study population n = 22
Control (n = 16)
P value
32.32 ± 3.98 21.36 ± 0.65 22.79 ± 1.51 10.09 ± 4.52 1.50 ± 0.81 2.88 ± 0.35 1.06 ± 0.12 95.94 ± 16.32 88.18 ± 20.82 84.65 ± 19.03 0.88 ± 0.17 1–5
31.63 ± 3.82 21.50 ± 0.81 22.64 ± 2.92 9.70 ± 3.36 1.85 ± 0.63 2.64 ± 0.51 1.05 ± 0.28 100.75 ± 10.01 92.69 ± 30.99 86.81 ± 31.01 0.64 ± 0.21 –
0.592 0.490 0.641 0.918 0.089 0.183 0.506 0.450 0.773 0.829 0.002
31.66 ± 27.27
–
Bold number indicates p b 0.05, in other words statistically significant. APCR: activated protein C resistance. AT III: antithrombin III.
Fig. 1. The relative mRNA expressions in patients with implantation failure group. Bars represent mRNA expressions normalized to GAPDH and compared to control group using the REST. *P b 0.05.
Microarray technology was used to evaluate gene expression variations during the transition period from pre-receptive phase to receptive phase on human endometrial samples. However; only a few genes were found to be similar in many studies whereas most studies indicated different regulatory pathways (Carson et al., 2002; Mirkin et al., 2005; Riesewijk et al., 2003; Talbi et al., 2006). According to our research, only one study showed that expression of these genes were up-regulated within the implantation window and MFAP-5 and NLF2 were proposed as new biomarkers for the exploration of endometrial receptivity (Haouzi et al., 2009a). Haouzi et al. (2009a) compared gene expression patterns on endometrial biopsy samples from 31 patients in a natural cycle preceding an ICSI procedure for severe male factor, taken 2 days after LH peak (LH + 2; early secretory phase) and 7 days after LH peak (LH + 7; midsecretory phase) and showed increased levels of expressions of 4 new markers including microfibril-associated protein 5 (MFAP5) and nuclear localized factor 2 (NLF2), which were not listed in previous microarray analyses (Haouzi et al., 2009a; Mirkin et al., 2005; Riesewijk et al., 2003). The other genes were laminin ß3, angiopoietin-like 1 (ANGPTL) and endocrine gland-derived vascular endothelial growth factor (EG-VEGF). NLF2 has been identified as a nuclear factor. Warton et al. (2004) proposed that this gene belonged to a novel gene family encoding nuclear factors with a role in regulating genes which control cellular architecture. Activation of these genes might increase vascular permeability in acute inflammation. This gene might also play an important role in endometrial remodeling since it is expressed within the implantation window. It was well known that invasion into the endometrial stroma, an important step for implantation, was facilitated by inflammation. Our study clearly demonstrated that NLF2 expression decreased in endometrium of infertile patients with implantation failure as compared to fertile controls. Since NLF2 is a member of a family of regulatory genes family that plays a role in endothelial cell inflammation, its decreased expression level may explain implantation failure in the infertile group. MFAP2 is a major antigen of elastine associated microfibrils and has been suggested to have a role in the etiology of hereditary connective tissue disorders (Lemaire et al., 2005). Expression of MAGPs stimulates vascular structural development and disorganized vascular structure can play a role in implantation failure. MFAP5 has a role in Notch signal activation pathway involved in vascularization during embryogenesis, development and normal homeostasis (Shawber et al., 2003). Increased expression of MFAP5 gene has been shown during receptive phase of a normal menstrual cycle (Haouzi et al., 2009a). It can be inferred that different expression levels of MFAP5 gene in the infertile group as compared to fertile group may explain a possible implantation defect. In a study by Haouzi et al. (2009a), MFAP5 and NLF2 were introduced as new genes which were involved in implantation. However, in our study only NLF2 decreased in patients with implantation
E. Turkyilmaz et al. / Gene 508 (2012) 140–143
failure. In our study although MFAP2 and MFAP5 gene expressions were also decreased in the implantation failure group as compared to the control group, these differences were not statistically significant, so can be concluded based on our findings that NLF2 could be the associated gene with implantation failure. The discrepancies among the results of the different studies involve many explanations. The population studied in Haouzi et al's paper included only male factor patients, implying that no infertility factors could be detected for women. However, only fertile patients represent ideal population, since female partners of male factor patients should still have factors limiting fertility. Another major disadvantage of Haouzi et al. was that histologic confirmation of the endometrium samples was not performed before gene expressions were evaluated. In our study, all endometrial samples were histologically evaluated and those samples that were in receptive phase were used for gene expression evaluation. Discrepancies in studies regarding gene expression patterns within the implantation window could have emerged due to insufficient number of subjects in our study population. In fact, majority of gene expression analyses in endometrial biopsy studies have inadequate number of patients. Decreased expression patterns of MFAP2 and MFAP5 in our study could have been significant if analysis were performed in a large group of patients was higher. Another possible explanation may be that sampling was performed within the implantation window but on a different histological date. Implantation window begins 6–8 days after ovulation and it continues approximately for 4 days (Wilcox et al., 1999). Other reasons could be the unavailability of a standard definition of implantation failure, application of different statistical methods in the study design and interpretation of the studies. In fact, similar disadvantages exist in various studies which explore gene expression profiles in implantation. Investigating global genes in a group of patients with implantation failure after multiple IVF cycles would be helpful in identifying common genes. However, there is only one recent study on a similar patient population. Koler et al. (2009) investigated up and down regulation patterns of 313 genes on endometrium samples of 4 patients with at least 3 implantation failures by using microarray technology. In this study, only endometrium of patients with progesterone levels higher than 20 ng/ml on the 21st day of the cycle was biopsied. It was found that 288 genes were up regulated and 25 genes were down regulated in the study population. However, MFAP2, MFAP5, and NLF2 were not involved in the list of genes that were up or down regulated in Koler's study. While Cyclin E2, SFRP1 and LEF1 were found to be down regulated in a group of multiple implantation failure, Slug and DKK1 were found to be up regulated. Down regulation of Cyclin E2 gene and up regulation of Slug gene were found to be estrogendependent (Koler et al., 2009). A possible defect of endometrial responsiveness in multiple implantation failure groups can be inferred from this result. Limited number of patients and inability to look for all gene expressions were the handicaps of the study by Koler et al. In several published studies, genomic analyses have been done to investigate endometrial receptivity process in natural cycles (Mirkin et al., 2005). However, endometrium under external hormonal stimulus may show a different gene expression pattern than that of a natural cycle. Ovarian stimulation could change gene expression pattern significantly (Horcajadas et al., 2005). Recently, a transcriptional acti-
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vation defect has been shown on endometrially expressed genes in controlled ovarian stimulation cycles with gonadotropin treatment (Haouzi et al. 2009b). 5. Conclusions NLF2 gene expression levels and differences in MFAP2 and MFAP5 gene expressions (albeit being insignificant) between infertile group and control group draw attention to a genetic basis under implantation failure. Future research in this area may pave the way for new clinical implications and improve fertility rates. References Albig, A.R., Becenti, D.J., Roy, T.G., Schiemann, W.P., 2008. Microfibril-associate glycoprotein-2 (MAGP-2) promotes angiogenic cell sprouting by blocking Notch signaling in endothelial cells. Microvasc. Res. 76 (1), 7–14. Aplin, J.D., 2000. The cell biological basis of human implantation. Baillieres Best Pract. Res. Clin. Obstet. Gynaecol. 14 (5), 757–764. Carson, D.D., et al., 2002. Changes in gene expression during early to mid-luteal (receptive phase) transition in human endometrium detected by highdensity microarray screening. Mol. Human Reprod. 8 (9), 871–879. Coutifaris, C., et al., 2004. NICHD National Cooperative Reproductive Medicine Network., 2004. Histological dating of timed endometrial biopsy tissue is not related to fertility status. Fertil. Steril. 82 (5), 1264–1272. Denker, H.W., 1993. Implantation: a cell biological paradox. J. Exp. Zool. 266 (6), 541–558. Haouzi, D., et al., 2009a. Identification of new biomarkers of human endometrial receptivity in the natural cycle. Hum. Reprod. 24 (1), 198–205. Haouzi, D., et al., 2009b. Gene expression profile of human endometrial receptivity: comparison between natural and stimulated cycles for the same patients. Hum. Reprod. 24 (6), 1436–1445. Horcajadas, J.A., et al., 2005. Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles. Mol. Hum. Reprod. 11 (3), 195–205. Koler, M., Achache, H., Tsafrir, A., Smith, Y., Revel, A., Reich, R., 2009. Disrupted gene pattern in patients with repeated in vitro fertilization (IVF) failure. Hum. Reprod. 24 (10), 2541–2548. Konac, E., Alp, E., Onen, H.I., Korucuoglu, U., Asyali Biri, A., Menevse, S., 2009. Matrix metalloproteinases (MMP-2 and ‐9), their tissue inhibitors (TIMP-2 and ‐3) and cell adhesion molecules (ICAM-1 and VCAM-1): a possible role in the endometrium of cases with unexplained infertility and implantation failure after IVF. Reprod. Biomed. Online 19 (3), 391–397. Lemaire, R., Korn, J.H., Shipley, J.M., Lafyatis, R., 2005. Increased expression of type I collagen induced by microfibril-associated glycoprotein 2: novel mechanistic insights into the molecular basis of dermal fibrosis in scleroderma. Arhritis Rheum. 52 (6), 1812–1823. Mirkin, S., et al., 2005. In search of candidate genes critically expressed in the human endometrium during the window of implantation. Hum. Reprod. 20 (8), 2104–2117. Miyamoto, A., Lau, R., Hein, P.W., Shipley, J.M., Weinmaster, G., 2006. Microfibrillar proteins MAGP-1 and MAGP-2 induce Notch I extracellular domain dissociation and receptor activation. J. Biol. Chem. 281 (15), 10089–10097. Noyes, R.W., Hertig, A.T., Rock, J., 1975. Dating the endometrial biopsy. Am. J. Obstet. Gynecol. 122 (2), 262–263. Riesewijk, A., et al., 2003. Gene expression profiling of human endometrial receptivity on days LH + 2 versus LH + 7 by microarray technology. Mol. Hum. Reprod. 9 (5), 253–264. Shawber, C.J., Das, I., Francisco, E., Kitajewski, J., 2003. Notch signaling in primary endothelial cells. Ann. N. Y. Acad. Sci. 995, 162–170. Talbi, S., et al., 2006. Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology 147 (3), 1097–1121. Warton, K., Foster, N.C., Gold, W.A., Stanley, K.K., 2004. A novel gene family induced by acute inflammation in endothelial cells. Gene 342 (1), 85–95. Wathen, N.C., Perry, L., Lilford, R.J., Chard, T., 1984. Interpretation of single progesterone measurement in diagnosis of anovulation and defective luteal phase: observations on analysis of the normal range. Br. Med. J. 288 (6410), 7–9. Wilcox, A.J., Baird, D.D., Weinberg, C.R., 1999. Time of implantation of the conceptus and loss of pregnancy. N. Engl. J. Med. 340 (23), 1796–1799.
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
NLF2 gene expression in the endometrium of patients with implantation failure after IVF treatment Esengul Turkyilmaz a, Haldun Guner a, Mehmet Erdem a, Ahmet Erdem a, Aydan Asyali Biri a, Ece Konac b,⁎, Ebru Alp b, Hacer Ilke Onen b, Sevda Menevse b a b
Department of Obstetrics and Gynecology, Faculty of Medicine, Gazi University, 06500 Besevler, Ankara, Turkey Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, 06500 Besevler, Ankara, Turkey
a r t i c l e
i n f o
Article history: Accepted 18 July 2012 Available online 3 August 2012 Keywords: Repeated implantation failure In vitro fertilization Gene expression NLF2 MFAP2 MFAP5
a b s t r a c t The aim of this study was to analyze the expression of microfibril-associated protein 2 (MFAP2), microfibril-associated protein 5 (MFAP5) and nuclear localized factor 2 (NLF2) genes in patients with repeated IVF failure and compare with fertile population. Total RNA was isolated from 38 patients (repeated implantation failure, group 1, n = 22; fertile patients, group 2, n = 16). mRNA expression levels were measured quantitatively using real-time polymerase chain reaction. Our results showed that mRNA expression of NLF2 significantly decreased in the infertility group as compared to control group (P = 0.023). In addition a marked decrease was observed in the expression of MFAP2 in women with repeated implantation failure. In conclusion, NLF2 gene expression levels and differences in MFAP2 and MFAP5 gene expressions (albeit being insignificant) between infertile group and control group draw attention to a genetic basis under implantation failure. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Endometrium becomes receptive to blastocyst implantation for a limited period called “the implantation window”, 6–8 days after ovulation and remains receptive for 4 days (Wilcox et al., 1999). This limitation in time leads to low implantation rates even in natural cycles. Despite many advances in assisted reproductive technologies (ART), low implantation rate is still the most important factor negatively affecting success rates. Successful implantation depends on synchronization between the developmental stages of an embryo itself and the complex molecular and cellular events induced by paracrine and autocrine regulators in endometrium within the implantation window. Endometrial biopsy and dating of the endometrium according to the classical criteria of Noyes were once used widely to ascertain that the ovulation has Abbreviations: ANGPTL, angiopoietin-like protein 1; APCR, activated protein C resistance; ART, assisted reproductive technology; DKK1, Dickkopf-related protein 1; ECM, extracellular matrix; EG-VEGF, endocrine gland-derived vascular endothelial growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HB-EGF, heparin-binding EGF-like growth factor; ICSI, intra cytoplasmic sperm injection; IL-1, interleukin-1; IL-11, interleukin-11; IVF, in vitro fertilization; LEF1, lymphoid enhancer-binding factor 1; LH, luteinizing hormone; LIF, leukemia inhibitory factor; MAGP2, microfibril-associated glycoprotein 2; MFAP2, microfibrillar-associated protein 2; MFAP5, microfibrillarassociated protein 5; NLF2, nuclear localized factor 2; PCR, polymerase chain reaction; QRT-PCR, quantitative real time PCR; SFRP1, secreted frizzled-related protein 1; TSH, thyroid-stimulating hormone; UPL, universal probe library. ⁎ Corresponding author. Tel.: +90 312 202 46 34; fax: +90 312 212 46 47. E-mail address: [email protected] (E. Konac). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.07.031
occurred as well as to ensure that endometrium is in proper histological synchronization (Noyes et al., 1975). However, it was found that ‘out of phase’ endometrium was also frequently encountered in endometrial biopsy of fertile as well as infertile women (Coutifaris et al., 2004). Advances in molecular biology provided additional information regarding the different expression patterns of many genes through out implantation window. Many molecules including integrins, mucins, growth factors (HB-EGF), cytokines (LIF, leptin, IL-1, IL-11), and lipids have recently been identified as important markers of implantation process. Expression profiles of many genes and their products were used as markers of endometrial receptivity and implantation with the help of microarray technology in endometrial biopsy specimens. In a recent study, microfibril-associated protein 5 (MFAP5) and nuclear localized factor 2 (NLF2) were suggested as new candidate genes for markers of human implantation in natural cycles (Haouzi et al., 2009a). MFAP5 gene, also called microfibril-associated glycoproteins 2 (MAGP2), encodes a microfibril-associated glycoprotein, which is a component of microfibrils, an important structural component of elastic tissues, such as vasculature. This protein is located in a position that would allow it to potentially modulate cell‐matrix interactions and participate in cell signaling pathways by interacting with both extracellular matrix (ECM), such as collagen and cell-associated proteins, such as integrins, (Lemaire et al., 2005; Miyamoto et al., 2006). It has also been suggested that MFAP5 has a role in “Notch signaling activation”; a pathway involved in vascularization during embryogenesis, development and normal homeostasis (Haouzi et al., 2009a). MAGP-2 promotes angiogenic cell sprouting by antagonizing
E. Turkyilmaz et al. / Gene 508 (2012) 140–143
“Notch signaling” pathways in endothelial cells in vitro (Albig et al., 2008). MFAP2 gene, also called MAGP1, is a regulator gene of bone remodeling and its absence is associated with osteopenia. NLF2 was identified as a nuclear factor and a member of a regulatory genes family that plays a role in endothelial cell inflammation. It has been suggested that NLF2 is probably a part of the signaling pathway causing changes in cell architecture and adhesion in endothelial cell inflammation (Warton et al., 2004). Recently it has been shown that the NLF2 gene was strongly expressed in the endometrium of luteal day LH + 7 samples, emphasizing its role in endometrium remodeling during implantation window (Haouzi et al., 2009a). Implantation remains a rate-limiting step for the success of the in vitro fertilization (IVF) treatments. No data exist, in our knowledge, regarding the role of MFAP5, MFAP2 and NLF2 gene expressions in patients with implantation failure after repeated IVF attempts. We aimed to compare the expression of these genes in patients with repeated IVF failure with normal fertile population. 2. Materials and methods 2.1. Study design, patient selection and tissue collection All patients in the study group had primary infertility and recurrent implantation failures after IVF. Implantation failure was defined as failure to conceive after one or more IVF attempts in which at least one good quality embryo was transferred. The Ethics Committee of Gazi University Faculty of Medicine approved study and informed consents were obtained from all subjects. All patients in the study group had patent fallopian tubes confirmed by hysterosalpingography or laparoscopy. Patients with unilateral tubal occlusion were included in the study group. Patients with factors or lesions that interfere negatively with implantation, such as severe male infertility and intracavitary lesions like polyps, fibroids, intrauterine adhesions and patients with uterine anomalies were excluded from the study group. Patients with endometriosis were excluded from the study. Patients with anovulation were also excluded since only endometrial biopsies in ovulatory patients can reveal secretory endometrial pattern. Control group was composed of fertile, ovulatory women who had at least one term live birth without any previous clinical pregnancy loss. Both groups were evaluated for thyroid functions and thrombophilic factors. Serum TSH levels were measured in all patients before recruiting them for the study. All women were tested for the presence of Factor V Leiden, prothrombin (G20210A) and methylenetetrahydrofolate reductase (C677T) mutations as well as for deficiencies in proteins S, C and antithrombin III and for resistance to activated protein C. Endometrial biopsy samples were obtained from the anterior wall of the uterine cavity using a “Pipelle catheter”. Samples were obtained during midsecretory phase‐day 7 in patients after endogenous luteinizing hormone (day LH + 7) peak or day 6 in patients after documentation of ovulation by ultrasonography. Serum progesterone levels were measured in all patients on the day of biopsy and patients with normal midluteal progesterone levels were included in the study. Any serum progesterone level greater than 6 ng/ml provides objective evidence that ovulation has occurred (Wathen et
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al., 1984). Histological evaluation of endometrial samples were made using the criteria described by Noyes et al. and only samples with normal maturation in relation to the corresponding cycle day were used for evaluation of gene expressions (Noyes et al., 1975). 2.2. Total RNA extraction and reverse transcription PCR (RT-PCR) Total RNA was isolated from endometrial tissue (approximately 50–100 mg) using the Trizol reagent (peqGOLD TriFast™, peqlab, Erlangen, Germany) as previously described by Konac et al. (2009). To avoid DNA contamination, we applied some modifications to manufacturer's instructions. RNA-containing pellet was treated with approximately 1–5 U RNase-free DNase per μg RNA and incubated at 37 °C for 30 min before being washed with 75% ethanol. The yield and quality of the RNA of each sample was determined by measuring the absorbance at 260 and 280 nm using the “NanoDrop spectrophotometer” (NanoDrop ND-1000, Montchanin, DE, USA). Total RNA (1 μg) was reverse-transcribed in a 20 μl reaction mixture using random hexamers and Transcriptor First Strand cDNA synthesis kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. 2.3. Quantitative real-time PCR (QRT-PCR) analysis MFAP2, MFAP5 and NLF2 mRNA expression levels were measured using the real-time PCR method. Probes and primer sets for each gene were designed at the ProbeFinder. Assay Design Center website (https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp? id=uplst 030000). PCR primers (exon–exon junction to allow discrimination between cDNA and gDNA) and Universal Probe Library (UPL) numbers for this study are provided in Table 1. The 10 μl reaction mixture, prepared in borosilicate glass capillaries, contained 1 × LightCycler TaqMan Master reaction mixture (Roche Diagnostics, GmbH, Mannheim, Germany), 2.5 pmol of each primer, 1 pmol of UPL probe, 4 mM MgCl2 and 1 μl cDNA. The real-time PCR assay included a no-template control. All PCR reactions were performed in the LightCycler 1.5 instrument (Roche Diagnostics, GmbH, Mannheim, Germany) using the following program conditions: 95 °C for 10 min followed by 45 cycles at 95 °C for 10 s , 60 °C for 20 s and finally a cooling step to 40 °C. We analyzed the expression of GAPDH housekeeping gene to normalize the results obtained by QRT-PCR. Each sample was tested in triplicate. PCR efficiency for each gene was tested by serial dilutions and amplification efficiencies of target genes and GAPDH were approximately equal. 2.4. Statistical analysis Mann–Whitney U test was performed using SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA) to compare both groups with regard to age, sampling day, endometrial date according to histological findings, serum progesterone and thyroid hormone levels and levels of acquired thrombophilia factors. Fisher's exact test and Chi-square test were used to compare both groups with regard to gene mutations resulting in thrombophilia. A P-valueb 0.05 was considered significant. All values were expressed as mean ± standard deviation (SD). Statistical
Table 1 The gene-specific primer sequences and probe numbers. Gene
Forward primer
Reverse primer
UPL probe no.
GAPDH MFAP2 MFAP5 NLF2
5′-AGCCACATCGCTCAGACAC-3′ 5′-TGCAAACAGTGTCTCAACGA-3′ 5′‐GGAACACGAAGCTATGAAAGATG-3′ 5′-GCTTGCAACCAGATCCAGAG-3′
5′-GCCCAATACGACCAAATCC-3′ 5′-CTTGTCCCGACAGAGGTCAG-3′ 5′-GGGTCTCTGCAAATCCACAT-3′ 5′-GGCCGAGGAACAGAGTTTC-3′
60 75 69 75
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significance levels of differences in mRNA expressions were analyzed by relative expression software tool (REST 2008 version 2.07). 3. Results All patients in the study group had primary infertility. Mean infertility duration was 8.8 ± 5.1 years (minimum 3; maximum 20 years). All patients had repeated IVF failure during a period of 1 to 5 years. 22 patients had unexplained infertility. Mean sperm count was 31.7 × 10 6 /mL. (± 27.3 × 10 6) (range 1–90 × 10 6) and percentage motility was 43.8 ± 17.4 (range 24%–43%). There was no difference among patients and controls in terms of age, sampling days, serum progesterone levels, thyroid function tests and thrombophilia parameters except for activated protein C resistance (P > 0.05). Detailed characteristics of the sample study population and the P-values are reported in Table 2. “Activated protein C” resistance (APCR) level was detected higher than normal level in 10 patients from the infertile group and 4 patients in the fertile group and the difference was statistically significant. Protein C, protein S and antithrombin III (AT III) values were not different between two groups. mRNA expression of MFAP2 was decreased in implantation failure group as compared to control group (1.34 fold change; P = 0.362). In contrast there was a 1.37-fold increase in the expression level of MFAP5 in the infertile patients with implantation failure as compared to control group (P = 0.138). The expression level of NLF2 decreased 1.84 folds in the implantation failure group as compared to the control group (P = 0.023). Comparison of MFAP2, MFAP5 and NLF2 gene expressions was demonstrated in Fig. 1. 4. Discussion In this study, expression profiles of MFAP5, MFAP2 and NLF2 genes were investigated in endometrial samples during the implantation window in a group of patients with implantation failure defined by multiple unsuccessful IVF attempts and the findings were compared with those of fertile controls. Our results showed that mRNA expression of NLF2 significantly decreased in the infertility group as compared to control group. The gene expression profiles of MFAP2 and MFAP5 were not different between infertile and fertile groups. Implantation is a process which involves attachment of the blastocyst to endometrium, creating an interaction between growing embryo and maternal circulation (Aplin 2000; Denker 1993). Many genes and molecules play significant roles in the implantation process. MFAP2, MFAP5 and NLF2 were recently recognized; thus their roles in the implantation process are yet to be determined. Table 2 Mean clinicopathologic characteristics of patients and controls.
Age Sampling day Histologic dating to Noyes criteria Serum progesterone (ng/ml) Serum TSH (mIU/L) Serum free T3 (pg/ml) Serum free T4 (ng/dl) AT III (%) Protein C (%) Protein S (%) APCR (%) No of previous in vitro fertilization cycles Sperm concentration (×106)
Study population n = 22
Control (n = 16)
P value
32.32 ± 3.98 21.36 ± 0.65 22.79 ± 1.51 10.09 ± 4.52 1.50 ± 0.81 2.88 ± 0.35 1.06 ± 0.12 95.94 ± 16.32 88.18 ± 20.82 84.65 ± 19.03 0.88 ± 0.17 1–5
31.63 ± 3.82 21.50 ± 0.81 22.64 ± 2.92 9.70 ± 3.36 1.85 ± 0.63 2.64 ± 0.51 1.05 ± 0.28 100.75 ± 10.01 92.69 ± 30.99 86.81 ± 31.01 0.64 ± 0.21 –
0.592 0.490 0.641 0.918 0.089 0.183 0.506 0.450 0.773 0.829 0.002
31.66 ± 27.27
–
Bold number indicates p b 0.05, in other words statistically significant. APCR: activated protein C resistance. AT III: antithrombin III.
Fig. 1. The relative mRNA expressions in patients with implantation failure group. Bars represent mRNA expressions normalized to GAPDH and compared to control group using the REST. *P b 0.05.
Microarray technology was used to evaluate gene expression variations during the transition period from pre-receptive phase to receptive phase on human endometrial samples. However; only a few genes were found to be similar in many studies whereas most studies indicated different regulatory pathways (Carson et al., 2002; Mirkin et al., 2005; Riesewijk et al., 2003; Talbi et al., 2006). According to our research, only one study showed that expression of these genes were up-regulated within the implantation window and MFAP-5 and NLF2 were proposed as new biomarkers for the exploration of endometrial receptivity (Haouzi et al., 2009a). Haouzi et al. (2009a) compared gene expression patterns on endometrial biopsy samples from 31 patients in a natural cycle preceding an ICSI procedure for severe male factor, taken 2 days after LH peak (LH + 2; early secretory phase) and 7 days after LH peak (LH + 7; midsecretory phase) and showed increased levels of expressions of 4 new markers including microfibril-associated protein 5 (MFAP5) and nuclear localized factor 2 (NLF2), which were not listed in previous microarray analyses (Haouzi et al., 2009a; Mirkin et al., 2005; Riesewijk et al., 2003). The other genes were laminin ß3, angiopoietin-like 1 (ANGPTL) and endocrine gland-derived vascular endothelial growth factor (EG-VEGF). NLF2 has been identified as a nuclear factor. Warton et al. (2004) proposed that this gene belonged to a novel gene family encoding nuclear factors with a role in regulating genes which control cellular architecture. Activation of these genes might increase vascular permeability in acute inflammation. This gene might also play an important role in endometrial remodeling since it is expressed within the implantation window. It was well known that invasion into the endometrial stroma, an important step for implantation, was facilitated by inflammation. Our study clearly demonstrated that NLF2 expression decreased in endometrium of infertile patients with implantation failure as compared to fertile controls. Since NLF2 is a member of a family of regulatory genes family that plays a role in endothelial cell inflammation, its decreased expression level may explain implantation failure in the infertile group. MFAP2 is a major antigen of elastine associated microfibrils and has been suggested to have a role in the etiology of hereditary connective tissue disorders (Lemaire et al., 2005). Expression of MAGPs stimulates vascular structural development and disorganized vascular structure can play a role in implantation failure. MFAP5 has a role in Notch signal activation pathway involved in vascularization during embryogenesis, development and normal homeostasis (Shawber et al., 2003). Increased expression of MFAP5 gene has been shown during receptive phase of a normal menstrual cycle (Haouzi et al., 2009a). It can be inferred that different expression levels of MFAP5 gene in the infertile group as compared to fertile group may explain a possible implantation defect. In a study by Haouzi et al. (2009a), MFAP5 and NLF2 were introduced as new genes which were involved in implantation. However, in our study only NLF2 decreased in patients with implantation
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failure. In our study although MFAP2 and MFAP5 gene expressions were also decreased in the implantation failure group as compared to the control group, these differences were not statistically significant, so can be concluded based on our findings that NLF2 could be the associated gene with implantation failure. The discrepancies among the results of the different studies involve many explanations. The population studied in Haouzi et al's paper included only male factor patients, implying that no infertility factors could be detected for women. However, only fertile patients represent ideal population, since female partners of male factor patients should still have factors limiting fertility. Another major disadvantage of Haouzi et al. was that histologic confirmation of the endometrium samples was not performed before gene expressions were evaluated. In our study, all endometrial samples were histologically evaluated and those samples that were in receptive phase were used for gene expression evaluation. Discrepancies in studies regarding gene expression patterns within the implantation window could have emerged due to insufficient number of subjects in our study population. In fact, majority of gene expression analyses in endometrial biopsy studies have inadequate number of patients. Decreased expression patterns of MFAP2 and MFAP5 in our study could have been significant if analysis were performed in a large group of patients was higher. Another possible explanation may be that sampling was performed within the implantation window but on a different histological date. Implantation window begins 6–8 days after ovulation and it continues approximately for 4 days (Wilcox et al., 1999). Other reasons could be the unavailability of a standard definition of implantation failure, application of different statistical methods in the study design and interpretation of the studies. In fact, similar disadvantages exist in various studies which explore gene expression profiles in implantation. Investigating global genes in a group of patients with implantation failure after multiple IVF cycles would be helpful in identifying common genes. However, there is only one recent study on a similar patient population. Koler et al. (2009) investigated up and down regulation patterns of 313 genes on endometrium samples of 4 patients with at least 3 implantation failures by using microarray technology. In this study, only endometrium of patients with progesterone levels higher than 20 ng/ml on the 21st day of the cycle was biopsied. It was found that 288 genes were up regulated and 25 genes were down regulated in the study population. However, MFAP2, MFAP5, and NLF2 were not involved in the list of genes that were up or down regulated in Koler's study. While Cyclin E2, SFRP1 and LEF1 were found to be down regulated in a group of multiple implantation failure, Slug and DKK1 were found to be up regulated. Down regulation of Cyclin E2 gene and up regulation of Slug gene were found to be estrogendependent (Koler et al., 2009). A possible defect of endometrial responsiveness in multiple implantation failure groups can be inferred from this result. Limited number of patients and inability to look for all gene expressions were the handicaps of the study by Koler et al. In several published studies, genomic analyses have been done to investigate endometrial receptivity process in natural cycles (Mirkin et al., 2005). However, endometrium under external hormonal stimulus may show a different gene expression pattern than that of a natural cycle. Ovarian stimulation could change gene expression pattern significantly (Horcajadas et al., 2005). Recently, a transcriptional acti-
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vation defect has been shown on endometrially expressed genes in controlled ovarian stimulation cycles with gonadotropin treatment (Haouzi et al. 2009b). 5. Conclusions NLF2 gene expression levels and differences in MFAP2 and MFAP5 gene expressions (albeit being insignificant) between infertile group and control group draw attention to a genetic basis under implantation failure. Future research in this area may pave the way for new clinical implications and improve fertility rates. References Albig, A.R., Becenti, D.J., Roy, T.G., Schiemann, W.P., 2008. Microfibril-associate glycoprotein-2 (MAGP-2) promotes angiogenic cell sprouting by blocking Notch signaling in endothelial cells. Microvasc. Res. 76 (1), 7–14. Aplin, J.D., 2000. The cell biological basis of human implantation. Baillieres Best Pract. Res. Clin. Obstet. Gynaecol. 14 (5), 757–764. Carson, D.D., et al., 2002. Changes in gene expression during early to mid-luteal (receptive phase) transition in human endometrium detected by highdensity microarray screening. Mol. Human Reprod. 8 (9), 871–879. Coutifaris, C., et al., 2004. NICHD National Cooperative Reproductive Medicine Network., 2004. Histological dating of timed endometrial biopsy tissue is not related to fertility status. Fertil. Steril. 82 (5), 1264–1272. Denker, H.W., 1993. Implantation: a cell biological paradox. J. Exp. Zool. 266 (6), 541–558. Haouzi, D., et al., 2009a. Identification of new biomarkers of human endometrial receptivity in the natural cycle. Hum. Reprod. 24 (1), 198–205. Haouzi, D., et al., 2009b. Gene expression profile of human endometrial receptivity: comparison between natural and stimulated cycles for the same patients. Hum. Reprod. 24 (6), 1436–1445. Horcajadas, J.A., et al., 2005. Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles. Mol. Hum. Reprod. 11 (3), 195–205. Koler, M., Achache, H., Tsafrir, A., Smith, Y., Revel, A., Reich, R., 2009. Disrupted gene pattern in patients with repeated in vitro fertilization (IVF) failure. Hum. Reprod. 24 (10), 2541–2548. Konac, E., Alp, E., Onen, H.I., Korucuoglu, U., Asyali Biri, A., Menevse, S., 2009. Matrix metalloproteinases (MMP-2 and ‐9), their tissue inhibitors (TIMP-2 and ‐3) and cell adhesion molecules (ICAM-1 and VCAM-1): a possible role in the endometrium of cases with unexplained infertility and implantation failure after IVF. Reprod. Biomed. Online 19 (3), 391–397. Lemaire, R., Korn, J.H., Shipley, J.M., Lafyatis, R., 2005. Increased expression of type I collagen induced by microfibril-associated glycoprotein 2: novel mechanistic insights into the molecular basis of dermal fibrosis in scleroderma. Arhritis Rheum. 52 (6), 1812–1823. Mirkin, S., et al., 2005. In search of candidate genes critically expressed in the human endometrium during the window of implantation. Hum. Reprod. 20 (8), 2104–2117. Miyamoto, A., Lau, R., Hein, P.W., Shipley, J.M., Weinmaster, G., 2006. Microfibrillar proteins MAGP-1 and MAGP-2 induce Notch I extracellular domain dissociation and receptor activation. J. Biol. Chem. 281 (15), 10089–10097. Noyes, R.W., Hertig, A.T., Rock, J., 1975. Dating the endometrial biopsy. Am. J. Obstet. Gynecol. 122 (2), 262–263. Riesewijk, A., et al., 2003. Gene expression profiling of human endometrial receptivity on days LH + 2 versus LH + 7 by microarray technology. Mol. Hum. Reprod. 9 (5), 253–264. Shawber, C.J., Das, I., Francisco, E., Kitajewski, J., 2003. Notch signaling in primary endothelial cells. Ann. N. Y. Acad. Sci. 995, 162–170. Talbi, S., et al., 2006. Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology 147 (3), 1097–1121. Warton, K., Foster, N.C., Gold, W.A., Stanley, K.K., 2004. A novel gene family induced by acute inflammation in endothelial cells. Gene 342 (1), 85–95. Wathen, N.C., Perry, L., Lilford, R.J., Chard, T., 1984. Interpretation of single progesterone measurement in diagnosis of anovulation and defective luteal phase: observations on analysis of the normal range. Br. Med. J. 288 (6410), 7–9. Wilcox, A.J., Baird, D.D., Weinberg, C.R., 1999. Time of implantation of the conceptus and loss of pregnancy. N. Engl. J. Med. 340 (23), 1796–1799.