Abstract STUDY QUESTION Does gene expression of putative endometrial implantation markers vary in expression between menstrual cycles? SUMMARY ANSWER In fertile women the expression of certain genes exhibits a pattern of stable regulation.which is not affected even when sampled twice in one cycle. WHAT IS KNOWN ALREADY Successful implantation occurs in a minority of IVF embryo transfers. In contrast to knowledge regarding the ovulatory process, there is a sparse understanding of endometrial genes critical to implantation. This lack of knowledge hinders progress in this field. STUDY DESIGN, SIZE, DURATION Endometrial pipelle samples were collected based on blood endocrinological markers at 2 and 7 days post initial LH surge. Five samples were collected over four cycles where the interval between collections ranged from sequential months to three years. PARTICIPANTS/MATERIALS, SETTING, METHODS Six fertile women attending an IVF clinic for male factor infertility, had samples collected. Global gene expression profiles were obtained from laser-microdissected, endometrial glands and stroma. Nineteen potential proliferation, cytokine and adhesion markers based on previous validated reports were studied. MAIN RESULTS AND THE ROLE OF CHANCE There was a significant modification between LH+2 and LH+7 of expression for 23 genes—11 in 8 in glands and stroma, 4 in stroma only and 3 in glands only suggesting stable, controlled regulation. Nevertheless, genes exhibited individual characteristics, e.g MKI67 exhibited lower expression at LH+7 than LH+2 and CCL4 higher, whereas TRO expressed limited difference in both cell types. Stability between cycles was demonstrated for gene expression at both LH+2—more than 60% of genes had <25% variation and at LH+7—60% had <30% variation. Further, effects of prior collection of an LH+2 sample on gene expression at LH+7 were not detected. The range of mRNA expression suggested that a clinical/diagnostic sample at LH+2 and LH+7 is likely to be a better index of endometrial function than a single sample. The possibility of redundancy suggests a panel would be more informative than a single marker. LARGE SCALE DATA Raw and normalized microarray data have been deposited with the EMBL’s European Genome-Phenome Archive for collaborative analysis, reference ega-box-815 (Lappalainen I, Almeida-King J, Kumanduri V, Senf A, Spalding JD, Ur-Rehman S, Saunders G, Kandasamy J, Caccamo M, Leinonen R et al. The European Genome-phenome Archive of human data consented for biomedical research. Nat Genet 2015;47:692–695.) [https://www.ebi.ac.uk/ega/home]. LIMITATIONS, REASONS FOR CAUTION This type of research has difficulties of recruitment of fertile women for multiple blood testing and repeat endometrial biopsies. Therefore, these data had decreased statistical power due to the overall participant numbers. However, the inclusion of four cycles for each participant permitted the aim of obtaining information on intercycle and intracycle variability to be achieved. WIDER IMPLICATIONS OF THE FINDINGS Our results support the feasibility of a clinical means of identification of a functional receptive endometrium. The robustness of data from individual women suggests that samples from one cycle can generally be applied to subsequent cycles. STUDY FUNDING/COMPETING INTEREST(S) Funding was granted from the Tertiary Education Commission of New Zealand, Contract I.D.:UOOX06007. There are no competing interests. microdissected endometrial glands and stroma, microarrays, mRNA expression, four cycles, intercycle/intracycle variation Introduction Implantation of the blastocyst into the uterine endometrium remains a significant limiting step for success in IVF methodologies (Dekel et al., 2014). However, we still lack an in depth understanding of endometrial physiology and its role in implantation (Clancy, 2009; Galliano et al., 2015). It is critical that we gain an understanding of the fertile/healthy endometrial physiology that can successfully support implantation and subsequent placentation before we attempt to unravel how perturbations of endometrial tissue lead to infertility. A number of factors have been investigated in search of endometrial markers of receptivity (Williams et al., 2001; Carson et al., 2002; Kao et al., 2002; Borthwick et al., 2003; Mirkin et al., 2005; Haouzi et al., 2009, Ordi et al., 2003; Riesewijk et al., 2003; Teh et al., 2016) but with limited indication that a specific key marker will define and be associated with a receptive window of implantation (WOI). With the introduction of profiling of the endometrium for the assessment of uterine receptivity, an endometrial receptivity array (ERA) has been proposed (Diaz-Gimeno et al., 2011). Other studies that used endometrial transcriptomics compared infertile women with others presumed to be fertile or participating as oocyte donors (Tapia-Pizarro et al., 2014; Garcia-Velasco et al., 2015; Altmae et al., 2016; Choi et al., 2016; Comstock et al., 2017, Huang et al., 2017; Mahajan, 2015). One aspect that is uncertain in fertile women is the ranges of levels of relevant indicators and their changes throughout a cycle. Additionally, limited studies of intercycle variation have failed to define the threshold of relevant indicators (Ordi et al., 2003; Clancy, 2009). Thus, there is a paucity of work published on which molecular changes must occur consistently to prepare a receptive endometrium. Studies of endometrial gene expression profiles and their influence toward successful implantation have been on-going for decades (Carson et al., 2002; Kao et al., 2002; Borthwick et al., 2003; Haouzi et al., 2009; Evans et al., 2012a, 2012b; Riesewijk et al., 2003, Mirkin et al., 2005). Most projects, focussed on the secretory phase of the endometrium when the tissue is most receptive to implantation by a blastocyst. However, there is an urgent need for information into endometrial variation which may influence implantation due to a key gap in current understanding which inhibits recognition of fertile, functional, healthy levels of relevant markers. The important value of this project is that it was performed on natural cycles without exogenous treatment interfering with physiological mechanisms. This baseline gene expression then offers comparison before testing manufactured cycles. In this study we have extended our investigations of the non-receptive LH+2 and mid-secretory, receptive LH+7 phase using our novel approach of laser microdissected endometrial glands and stroma to investigate differences in mRNA expression profiles to ascertain the extent of cycle variability. This methodological approach differs from previous studies of undifferentiated tissue. Our sample set constituted a well-defined fertile phenotype where samples were collected based on blood endocrinological markers and confirmed by histological characteristics (Evans et al., 2012a, 2012b). The two time points were chosen as Ova Pick Up (OPU) is performed at the non-receptive LH+2 and used as a reference baseline, whereas at LH+7 the embryo is transferred back into the uterus when the endometrium is considered to be receptive. Previously reported markers associated with proliferation, cytokine activity and cell adhesion were investigated. Cycle to cycle variability both between and within individuals was investigated in fertile women during natural cycles to determine endometrial stability. We identified variation in fertile women using our database of global mRNA expression that profiles the fertile endometrium. This information will build a foundation of understanding (Clancy, 2009) and act as a reference to assist subsequent study of the unexplained reasons for failed IVF where endometrial inadequacy is a contributing factor. Materials and Methods This project received the approval of the Upper South A Ethics Committee, New Zealand, application URA/07/03/021. Participants gave written consent and sampling was performed at a single IVF clinic. Participants Six Caucasian women with no uterine pathology nor any confounding medical condition and with partners with male factor infertility were recruited. All the women had proven fertility and conceived without delay using ICSI and subsequently delivered a live birth ≤3 years before this study. The women had a BMI < 30 [mean ± SEM; 24.9 ± 0.9], aged 20–40 years [mean ± SEM; 36.5 ± 0.9], and had regular menstrual cycles with proven ovulation, were non-smokers and were taking no medication. Cycle length ranged from 21–29 days [mean 26.4 days]. Each woman had samples collected by pipelle biopsy at days defined by the rise in blood LH concentrations (WHO Task Force Investigators, 1980) when the LH level had doubled in concentration from the baseline above 15 IU/l. Samples were collected two days post first significant surge [LH+2] and seven days post first significant surge [LH+7] in natural cycles. From each participant, five samples were obtained over four cycles; in one cycle only an LH+2 sample was taken, in two cycles only an LH+7 sample and in the remaining cycle both a LH+2 and a LH+7 sample. Three of the women had samples collected in consecutive months [sample set numbers 2,3,9], The other three women had samples collected over some years [sample set numbers 7,10,23], with one woman  having a baby between sample collections. Sampling was performed with every attempt made to collect samples in the same order, however, this was not always possible. Every attempt was also made to sample from the same site. Endometrial sampling was from uterine fundus using a standard endometrial suction curette [Pipelle de Cornier, Laboratoire C.C.D. Paris, France] as described (Polena et al., 2007). Sample collection and processing Cycle monitoring: Blood samples were collected daily near mid-cycle to establish the first significant rise in LH concentration, the best hormonal predictor of ovulation (WHO Task Force Investigators, 1980). At this time, hormone concentrations in blood were measured. Oestradiol ranged from 400–2000 pmol/l [mean 1060 pmol/l]; progesterone 2–4 nmol/l [mean 3 nm/l]; FSH levels ranged from 4–31 IU/l [mean 14.1 IU/l] and LH 12–81 IU/l [mean 39 IU/l]. The endometrial biopsy was cleaned of blood and mucus and then snap frozen in liquid nitrogen [LN2] and stored at −80°C. The tissue samples used in this study were also dated by histological evaluation using our published parameters (Evans et al., 2012a, 2012b). Tissue processing was as described previously (Evans et al., 2012a, 2012b). Briefly, total RNA was extracted from the separate laser microdissected glandular and stromal cellular components using PicoPure columns [Arcturus]. Linear PCR was performed on five microliters of mRNA with NuGen WT-Ovation FFPE RNA amplification system [NuGen Technologies, USA] producing single stranded [ss]cDNA. Fragmentation and labelling of 5 μg of sscDNA was performed using NuGen FL-Ovation cDNA Biotin module [NuGen Technologies, USA]. The resultant product was hybridized to Affymetrix Human Genome [HG] U133 Plus 2.0 arrays [manufacturer’s protocol; Affymetrix, USA] Arrays were scanned on a GeneChip® Scanner 3000 7 G using the Affymetrix GeneChip® Command Console® Software [AGCC]. Microarray analysis Based on the analysis methods described previously (Evans et al., 2012a, 2012b), the statistical environment ‘R’ (RDevelopmentCoreTeam, 2010) and contributed packages from the R software repository Bioconductor [http://www.bioconductor.org/] were used for data analysis. First, quality control was performed (Parman and Halling, 2006; Kauffmann et al., 2009) before quantile normalization using the Robust Multichip Averaging (RMA) algorithm with background correction from the R ‘affy’ package (Gautier et al., 2004). Statistical testing was performed using t-tests as well as ‘LIMMA’ (Smyth, 2004) and ‘equivalence’ packages (Robinson, 2016). Variance components were estimated using the varComp function of the ‘nlme’ package (Pinheiro et al., 2017). Raw and normalized microarray data have been deposited with the EMBL’s European Genome-Phenome Archive for collaborative analysis, reference ega-box-815 (Lappalainen et al., 2015) [https://www.ebi.ac.uk/ega/home]. This data set provided an enormous amount of information of which this first project is just the beginning of these investigations. mRNA markers The experimental plan is illustrated in Fig. 1. Figure 1 View largeDownload slide An outline of the comparisons made to determine expression of marker genes within and between fertile cycles. (1) Stability of expression of endometrial mRNAs in the LH+2 biopsies from the same woman sampled in two separate cycles. (2) Comparison of expression of endometrial mRNAs in the LH+7 biopsies (a) cycles without a prior biopsy at LH+2, (b) with or without a prior biopsy at LH+2 in the same cycle and (c) all three cycles. (3) Comparison of expression of endometrial mRNAs in samples at LH+2 and LH+7, (a) all three LH+7 cycles (b) cycles without or (c) cycles with a prior biopsy at LH+2 in the same cycle. Figure 1 View largeDownload slide An outline of the comparisons made to determine expression of marker genes within and between fertile cycles. (1) Stability of expression of endometrial mRNAs in the LH+2 biopsies from the same woman sampled in two separate cycles. (2) Comparison of expression of endometrial mRNAs in the LH+7 biopsies (a) cycles without a prior biopsy at LH+2, (b) with or without a prior biopsy at LH+2 in the same cycle and (c) all three cycles. (3) Comparison of expression of endometrial mRNAs in samples at LH+2 and LH+7, (a) all three LH+7 cycles (b) cycles without or (c) cycles with a prior biopsy at LH+2 in the same cycle. Investigations into whether there is variation or tight functional linkage between chosen mRNAs and the endocrinological factors that are part of the implantation process were performed. Comparative expression of a selected set of markers were analysed (Fig. 1). Where several probes for some of the mRNAs occurred the expression provided by one probe per mRNA was analysed. Specific markers of mRNA expression were chosen for analysis by noting previously proposed candidates in samples that had been independently validated in other relevant studies; 1. Proliferation, 2. Cytokine activity and 3. Adhesion: Proliferation markers: Proliferating cell nuclear antibody [PCNA] (Niklaus et al., 2007; Evans et al., 2012a, 2012b), Marker of KI67 [MKI67] (Niklaus et al., 2007; Kuokkanen et al., 2010) (Evans G.E.et al., unpublished data), Cyclin A2 [CCNA2] (Niklaus et al., 2007; Evans et al., 2012a, 2012b), Mini-chromosome maintenance 2 [MCM2] (Niklaus et al., 2007; Kuokkanen et al., 2010) and Mothers against decapentaplegic homologue 3 [SMAD3] (Evans et al., 2012a, 2012b). Cytokine markers: Interleukin 11 [IL11] (van Mourik et al., 2009; Dimitriadis et al., 2010), Leukaemia inhibitory factor [LIF] (van Mourik et al., 2009; Dimitriadis et al., 2010, Rosario and Stewart, 2016), Chemokine [C-X3-C motif] ligand 1 [CX3CL1] (van Mourik et al., 2009; Dimitriadis et al., 2010), Chemokine [C-C motif] ligand 4 [CCL4] (van Mourik et al., 2009; Dimitriadis et al., 2010; Gnainsky et al., 2015), Chemokine [C-C motif] ligand 19 [CCL19] in mouse (Guerin et al., 2011) and Interleukin 15 [IL15] (Gnainsky et al., 2010), (Evans G.E. et al., unpublished data). Adhesion markers: E-cadherin [CDH1] (Rahnama et al., 2009; van Mourik et al., 2009; Yang et al., 2017), Protocadherin-1 [PCDH1] (Sherwin et al., 2008), Trophinin [TRO] (Tabibzadeh, 1998; Armant, 2011; Fukuda and Sugihara, 2012), Tastin [TROAP] (Tabibzadeh, 1998; Armant, 2011; Fukuda and Sugihara, 2012) (Evans G.E. et al., unpublished data), Bystin [BYSL] (Armant, 2011; Fukuda and Sugihara, 2012), Integrin α5 [ITGA5] (Ordi et al., 2002), Integrin β3 [ITGB3] (Ordi et al., 2002; Gnainsky et al., 2015) and Integrin β1 [ITGB1](van Mourik et al., 2009; Gnainsky et al., 2015). Data characteristics and analysis The raw data are collected in log2 transformed format and statistical analyses were performed on linearized values. LH+2 biopsy in one cycle compared to LH+2 biopsy in another cycle in the same woman The expression of the mRNAs was determined for both LH+2 cycles in each of the six women. It was assumed that no systematic change was brought about by previous sampling; whether a sample was in one set or the other would be a random occurrence. The results will give an indication of overall stability of expression at LH+2 of the mRNA markers. The levels of markers detected in the two cycles were compared using a paired t-test. For each woman the mean of expression of the two cycles was calculated, percentage variation was used as the index of variation of expression and was defined as 100 times the absolute difference between a LH+2 value and the mean LH+2 value divided by the mean. Variation in expression at LH+7 There were three analyses performed and described as follows. LH+7 biopsy without a prior biopsy in one cycle compared to LH+7 without a prior biopsy in another cycle in the same woman The analysis was performed similarly to that for the LH+2 sample (see section Data characteristics and analysis). Variation of the ratio of LH+7/LH+7 in individual women without prior biopsy were calculated. The mean level of a marker at the first LH+7 biopsy and that at the other LH+7 biopsy was calculated. The absolute difference of expression at each time from the mean was calculated and its percent of the mean determined. The mean percent difference for a marker for each of the six women provided the index of variation of expression for that marker. Whether the values were different was determined using a paired t-test [paired for each woman] for each marker. LH+7 biopsy in cycles without an LH+2 biopsy compared to LH+7 biopsy in another cycle with an LH+2 biopsy in the same woman To determine if the prior biopsy has a systematic effect on mRNA expression, for each woman the expression of a marker in the two samples from cycles without a prior biopsy were averaged and compared to the expression in the sample taken following a biopsy at LH+2. A paired t-test was used, investigating the hypothesis that a prior biopsy would modify expression of the putatively implantation-related mRNA. All LH+7 biopsies with and without a prior LH+2 biopsy. The average absolute difference of expression from the mean for each of the three biopsies taken at LH+7, including the two samples without prior biopsy and the one sample with prior biopsy were divided by the mean expression of a marker and converting the fraction to a percentage. The mean percent variations for the level of a marker in the group of women was determined. Variation in mRNA expression between LH+2 and LH+7 Of particular interest in this project was the way the endocrinological and metabolic preparation for implantation might occur by the regulation of mRNAs between LH+2 and LH+7. The change was investigated across cycles and within a cycle as described as follows. LH+2 biopsy in one cycle compared to LH+7 biopsy in separate cycles in the same woman The ratio of expression of LH+7 and LH+2 was calculated for biopsies taken in separate cycles in the same woman, where two biopsies at LH+2 and the two samples at LH+7 taken in separate cycles were incorporated into the calculation. LH+2 biopsy in one cycle compared to LH+7 biopsy in the same cycle in the same woman Further details of the relationship between expression at LH+7 and LH+2 were investigated by considering only biopsies at both times [LH+2 and LH+7] taken during the same cycle. A paired t-test was used to allow for a range of expression in women across the group. The calculations of variation used absolute differences [rather than positive or negative] and thus allowed an assessment of variations from the mean in either direction and avoided potentially equalizing a wide variation to zero. LH+2 in all cycles and all women compared to LH+7 in all cycles and all women The expression exhibited by a marker in all the participating women at LH+2 and at LH+7 were pooled. A student’s t-test was employed to obtain an indication of whether there was a change in average expression for a mRNA between the two days. The assigned SEM is the sum of the SEMs of the LH+2 and LH+7 groups divided by the square root of two [n = 2]. Results We selected potentially indicative markers involved in: proliferation [i.e. PCNA, MKI67, CCNA2, MCM2 and SMAD3], cytokine markers [i.e. CX3CL1, CCL4, CCL19 and IL15] and adhesion markers [i.e. CDH1, PCDH1, TRO, TROAP, BYSL, ITGA5, ITGB3 and ITGB1]. Summary of data characteristics First, the contribution of the various experimental factors to overall mRNA expression of the entire microarray data set was estimated using the varComp function. These results suggested that approximately 21% of the overall variance was contributed by differences between gland and stromal mRNA expression, approximately 5% by differences between LH+2 and LH+7, approximately <2.0% by the cycle in which the sample was taken and by the identity of the participant. The other contributions to variance were interactions between these experimental factors and the residual. This suggests that the systematic differences between endometrial mRNA expression of different fertile women may be small relative to differences between LH+2 and LH+7. Data analysis All samples were processed and RNA extracted using identical methods and hybridized to microarrays in three batches. We saw no systematic RNA quality or transcriptome-wide RNA expression profile differences associated with: anatomical site [glands versus stroma], time in cycle, or cycle number. However, we did note that the majority of samples from two specific participants had globally lower probe-level signal intensities than the samples from other participants. After data normalization and summarization, differences attributable to these samples accounted for ~10% of overall data variance [based on Principle Component Variance Component analysis]. These differences were no longer visible in histograms of data after normalization. Those samples from these two participants were not enriched onto any side of the comparisons made in this study, and all conclusions drawn for this study were repeatable on re-analysis of the data with these samples excluded. Our analysis focussed on the intercycle consistency and intracycle regulation of mRNA expression profiles within individual women. An example of variations of expression are demonstrated in Fig. 2 noting: Figure 2 View largeDownload slide Examples of a range of patterns of mRNA regulation in six individual women in chronological sequence of biopsies collected. (A) downregulation of a marker (MKI67) at LH+7 compared to its expression at LH+2 in glands and stroma. (B) upregulation of a marker (CCL4) at LH+7 compared to its expression at LH+2 in glands and stroma (C) no regulation of a marker (SMAD3) in stroma but upregulation in glands at LH+7 compared to its expression on LH+2. (D) limited regulation of a marker (TRO) between LH+2 and LH+7. Contiguous time-points are linked in the graphs to highlight the variation in the cyclicity of the mRNA expression between LH+2 and LH+7. Note that not all cycles so joined are sequential. There is no line where two samples are not in the sequence of LH+2 then LH+7 or vice versa. Figure 2 View largeDownload slide Examples of a range of patterns of mRNA regulation in six individual women in chronological sequence of biopsies collected. (A) downregulation of a marker (MKI67) at LH+7 compared to its expression at LH+2 in glands and stroma. (B) upregulation of a marker (CCL4) at LH+7 compared to its expression at LH+2 in glands and stroma (C) no regulation of a marker (SMAD3) in stroma but upregulation in glands at LH+7 compared to its expression on LH+2. (D) limited regulation of a marker (TRO) between LH+2 and LH+7. Contiguous time-points are linked in the graphs to highlight the variation in the cyclicity of the mRNA expression between LH+2 and LH+7. Note that not all cycles so joined are sequential. There is no line where two samples are not in the sequence of LH+2 then LH+7 or vice versa. (A) down-regulation between LH+2 and LH+7 in both glands and stroma, (B) up-regulation (C) up-regulation in glands only and (D) limited regulation. Expression of markers are in chronological sequence of biopsy collection for the six individual women. Change between mRNA expression in samples taken at LH+2 in separate cycles This project was designed to establish a data set on ranges of endometrial mRNAs that are empirically present in fertile women. Our summary is that some mRNAs are more sensitive than others to the circumstances of sampling. Endometrial mRNAs observed at LH+2 in the same woman sampled in separate cycles were mainly equivalent in abundance. The expression in the first biopsies were compared against the second using a paired t-test. The exceptions were PCNA, MKI67 and MCM2, which were expressed significantly differently in the glands of the second LH+2 sample compared to the first LH+2 sample demonstrating biological variation. Overall, of the 38 markers [19 in each glands and stroma], approximately 2/3 [63%] had less than 25% variation between samples averaged across women (Fig. 3). The most variable markers were proliferation markers in glands with a mean variation of 38%. In all three groups of markers, the variation observed was higher in glandular tissue than stroma. For proliferation markers, mean ± SEM of the variation in stromal cells was 22.4% ± 1.6% and in glands 37.9% ± 3.3%; for cytokine markers, stroma 19.1% ± 4.5%, glands 23.4% ± 2.6%, for adhesion markers, stroma 22.7% ± 3.2%, glands 24.7% ± 2.8%. Figure 3 View largeDownload slide % variation of mRNA expression between LH+2 measurements made in separate cycles. Variation was calculated for each woman as 100 × absolute difference between a LH+2 value and the mean of the two LH+2 values divided by the mean. Data are mean ± SEM, n = 6. Figure 3 View largeDownload slide % variation of mRNA expression between LH+2 measurements made in separate cycles. Variation was calculated for each woman as 100 × absolute difference between a LH+2 value and the mean of the two LH+2 values divided by the mean. Data are mean ± SEM, n = 6. Change between mRNA expression in samples taken at LH+7 in separate cycles without a prior biopsy There were no statistically different endometrial mRNAs expressed in the endometrium of the same woman sampled in separate cycles at LH+7. Figure 4 depicts the ratio of one LH+7 sample to the other LH+7 and all ratios are approximately one indicating no robustly detectable difference. Figure 4 View largeDownload slide Ratios of mRNA expression between the first and second LH+7 cycles where samples were taken without a prior LH+2 biopsy. Data are mean ± SEM n = 6. Figure 4 View largeDownload slide Ratios of mRNA expression between the first and second LH+7 cycles where samples were taken without a prior LH+2 biopsy. Data are mean ± SEM n = 6. The mean mRNA expression in samples in LH+7 samples in cycles with and without a prior sample taken at LH+2 The potential effect of a prior pipelle biopsy was investigated. Some endometrial mRNAs were differently expressed in LH+7 samples in a cycle without prior biopsy compared to samples at LH+7 from women who had a pipelle biopsy at LH+2 in the same cycle 5 days prior (Fig. 5): The proliferation markers were not affected at all by prior sampling in the same cycle when compared to sampling in a separate cycle. CCL19 was the only cytokine marker in this group to be altered by sampling in the same cycle with expression in the stroma being lower in the LH+7 sample taken as the second sample of the cycle. ITGB1 was the only adhesion marker in this group with altered expression in the stroma which was lower in the LH+7 sample taken as the second sample of the cycle. Figure 5 View largeDownload slide Ratio of mRNA expression in LH+7 samples with a prior biopsy at LH+2 over those without a prior biopsy on LH+2. Diamonds indicate markers that had a significant difference of expression between the conditions: CCL19 and ITGB1 in stroma are indicated in yellow P ≤ 0.05. Figure 5 View largeDownload slide Ratio of mRNA expression in LH+7 samples with a prior biopsy at LH+2 over those without a prior biopsy on LH+2. Diamonds indicate markers that had a significant difference of expression between the conditions: CCL19 and ITGB1 in stroma are indicated in yellow P ≤ 0.05. The ratio of expression between the levels collected in distinct cycle conditions was predominantly close to 1, indicating approximately equivalent expression (Fig. 5) demonstrating the stability of expression of the functionalis endometria. Variation of mRNA expression at LH+7 The variation in expression of a marker in all three samples for a woman sampled at LH+7 was determined and the mean percent variations for a marker in the group of women was then depicted (Fig. 6). Most markers [22 of 38] [58%] had a variation that was less than 30% and all were less than 40% except for LIF and CDH1 in stroma and LIF in glands. Figure 6 View largeDownload slide % variation of mRNA expression at LH+7 of all three samples for a woman. Variation was calculated for each woman as 100 × absolute difference between a LH+7 value and the mean of the three LH+7 values divided by the mean. Data are mean ± SEM, n = 6. Figure 6 View largeDownload slide % variation of mRNA expression at LH+7 of all three samples for a woman. Variation was calculated for each woman as 100 × absolute difference between a LH+7 value and the mean of the three LH+7 values divided by the mean. Data are mean ± SEM, n = 6. Change between mRNA expression in samples taken at LH+2 and LH+7 Change between mRNA expression in samples taken at LH+2 and LH+7 in separate cycles Some endometrial mRNAs exhibited differential expression when collected in separate cycles using a students t-test. The ratios of LH+2 to LH+7 collected in separate cycles are depicted in Fig. 7A: The proliferation markers PCNA, MKI67 and CCNA2 were all significantly down regulated at LH+7 compared to LH+2 in both glands and stroma, while MCM2 was down regulated in glands only. Conversely, SMAD3 was significantly upregulated at LH+7 in glands but not the stroma. These results are consistent with our previous observations (Evans et al., 2012a, 2012b). The results of MCM2 and SMAD3 expression demonstrate the potential for proliferation in glands to be distinctly regulated from that in stroma in response to the changing, cycling milieu of the endometrium. The cytokine marker, IL15 was significantly upregulated at LH+7 compared to LH+2 in both cell types, whereas, CCL4 was significantly upregulated in the glands only. The adhesion markers, CDH1and ITGA5 were significantly upregulated at LH+7 compared to LH+2 in both cell types, while PCDH1 was significantly upregulated in stroma only. ITGB1 was significantly downregulated at LH+7 compared to LH+2 in both cell types, while ITGB3 was downregulated at LH+7 in stroma only. This set of results indicates that different mRNAs are apparently independently regulated to provide an environment compatible with establishment of a pregnancy. Figure 7 View largeDownload slide Ratio of mRNA expression between LH+7 and LH+2 (A) collected in separate cycles, (B) collected in the same cycle and (C) in all biopsies collected. The results for the entire panel of 38 markers is presented. Those markers with a ratio less than 1 have higher expression at LH+2 and vice versa. The P-values that designate probable difference from unity are indicated: red circle, P < 0.001; green square, P < 0.01; yellow diamond, P < 0.05. Figure 7 View largeDownload slide Ratio of mRNA expression between LH+7 and LH+2 (A) collected in separate cycles, (B) collected in the same cycle and (C) in all biopsies collected. The results for the entire panel of 38 markers is presented. Those markers with a ratio less than 1 have higher expression at LH+2 and vice versa. The P-values that designate probable difference from unity are indicated: red circle, P < 0.001; green square, P < 0.01; yellow diamond, P < 0.05. Change between mRNA expression in samples taken at LH+2 and LH+7 in the same cycle Some endometrial mRNAs were differentially expressed between LH+2 and LH+7 from the same woman sampled in the same cycle 5 days apart using a paired t-test. The ratios of LH+2 to LH+7 in the same cycle are depicted in Fig. 7B: The proliferation markers were expressed in the same manner as in separate cycles, PCNA, MKI67 and CCNA2, were all significantly down regulated in both cell types, all as reported previously by this group (Evans et al., 2012a, 2012b). MCM2 was also significantly downregulated in both glands and stroma. Whereas SMAD3 was not significantly regulated. These findings differ from when samples were taken in separate cycles. mRNA expression at LH+7 was consistently lower than LH+2 for the proliferation markers PCNA, MKI67 and CCNA2 in the same cycle and in a separate cycle. This consistency strongly suggests that there was stability of function by the translated proteins. The cytokine marker CCL4 was significantly upregulated in both glands and stroma which differed from the result of sampling in separate cycles where the glands only were significantly regulated. Interestingly, IL11 was upregulated in the glands, a finding that differed from sampling in separate cycles where no regulation was reported. Further, IL15 was significantly upregulated in the stroma only which also differed from expression in sampling in separate cycles where both cell types were regulated. The adhesion markers were also affected by sampling in the same cycle as has been demonstrated by there being a loss of significant regulation of expression of PCDH1, TRO, TROAP, ITGB1 and ITGB3 in stroma and the loss of significant regulation of ITGA5 expression in glands. Overall ratio of mRNA expression at LH+7 from all three biopsies compared to expression of both biopsies at LH+2 The ratios of expressions at LH+7 to that at LH+2 in all biopsies were determined (Fig. 7C): Of the proliferation markers those that expressed significant differences between LH+2 and LH+7 in both glands and stroma were: PCNA, MKI67, CCNA2, MCM2 which were down-regulated; and in glands only; SMAD3 was up-regulated. Of the cytokine markers those that expressed significant differences in both glands and stroma were: IL15 and CCL4 which were up-regulated; and in glands only LIF was also up-regulated. Of the adhesion markers those that expressed significant difference in both glands and stroma were: ITGA5 was up-regulated and TROAP was down-regulated; in stroma only; PCDH1 was up-regulated, TRO, TROAP, ITGB1 and ITGB3 were both down-regulated in stroma only: CDH1 was up-regulated in glands only.To investigate the stability of expression, further analyses were performed. First, the ratios of expression at LH+7 to LH+2 taken in separate cycles (Fig. 7A) were compared to the statistical relationship from that obtained from all biopsies (Fig. 7C). There was a change in regulation of only 6 of the 38 markers [16%]. Second the ratios of expression at LH+7 compared to LH+2 taken in which biopsies were performed where both sampling times were within the same cycle (Fig. 7B) were compared to the statistical relationship from that obtained from all biopsies (Fig. 7C). The expression of proliferation markers was similar to that obtained from all biopsies except for SMAD3 in glands. For cytokine markers, the exceptions were IL11, LIF and IL15 which had a different level of significance in glands. The adhesion markers indicated more sensitivity to a prior sample taken in the same cycle where a different level of statistical significance in the regulation of these markers was noted. Almost half of the ratios of expression of these markers had altered levels of significance, being 7 changes in 16 [44%] markers. Thus, ITGB1 had variation noted in glands and stroma, while PCDH1, TRO, TROAP and ITGB3 had variation noted in stroma only and ITGA5 in glands only. Thus overall stability was demonstrated except for adhesion markers in stroma, which exhibited a more responsive sensitivity to prior sampling in the same cycle. Data of 38 markers from 6 women each with 5 endometrial biopsies The data from 38 selected markers are depicted in log2 format in Supplementary Fig. S1A–C. The mRNA expression of each marker is presented in chronological sequence in which the biopsies were collected from each of the six women. Contiguous time points are linked in the graphs. The range of levels of expression between women and between markers is depicted. The different patterns of regulation can be noted. Discussion The rationale for this work was to assess the endometrium of fertile women to better understand the events of the natural ovulatory cycle particularly at the time leading up to a state of endometrial receptivity. This report may assist in predicting if one cycle will have a better outcome than another when undertaking IVF. We previously reported the change in mRNA expression of cell cycle markers between the non-receptive and the receptive endometrial glands and stroma (Evans et al., 2012a, 2012b) and luminal epithelia (Evans et al., 2014) of fertile women in two separate natural cycles thereby establishing a reference database. We have now revisited that data and analysed data from other samples of glands and stroma to ascertain the changes in expression of a biologically relevant panel of mRNAs within a cycle in a woman. One of the limitations of this type of research includes difficulties of recruitment of fertile women to undergo the necessary blood endocrine testing and repeat endometrial biopsies, as noted previously. Nevertheless, adherence to the strict criteria of a healthy fertile phenotype provided a rare sample set although this study was limited to six participants. Additionally, the inclusion of four cycles for each participant and the focus on a small biologically relevant panel of previously validated mRNAs allowed the aim of obtaining information on intercycle and intracycle variability to be achieved. It was noted that one woman with consecutive sampling experienced shortening of cycle length from 27 to 21 days possibly due to this sampling process. Despite this effect she still exhibited the same pattern of mRNA expression as the other women. Although sample collection was completed over up to three years there was no detectable difference in the levels of mRNA expression between these women. Other gene expression methods, such as Endometrial Receptivity Array (ERA) (Diaz-Gimeno et al., 2011), aim to identify endometrial receptivity adequacy. This study will complement and expand such methodologies that are able to detect a dysregulated WOI in participants with unexplained infertility or recurrent implantation failure. Interestingly, of the list of 238 genes detected in multi-cellular tissue used by ERA to predict a receptive endometrium (Diaz-Gimeno et al., 2011) our previous data was able to confirm only 57 genes that were significantly expressed in microdissected glands and stroma. This is an example of the difference in results obtained by testing specific cell types compared to results previously reported on undifferentiated tissue (Carson et al., 2002; Kao et al., 2002; Borthwick et al., 2003; Haouzi et al., 2009; Riesewijk et al., 2003, Mirkin et al., 2005). Our use of microdissected tissue may also explain why more than 60% of the genes that we noted that were significantly up or down-regulated in glands and stroma between LH+2 and LH+7 have not been previously reported by other groups using undifferentiated tissue (Evans et al., 2012a, 2012b). Thus comparing the results from these works is difficult partly because methodology and microarray platforms differ. Further, our earlier work using dual platform confirmation found only six Affymetrix and five Agilent probes that were not detected by the other platform. We have addressed the important question regarding the possible intercycle variability or consistency of an endometrial genomic signature in fertile women. This proof of principle work was based on previously reported markers only, which may have restricted this current work and other more decisive markers may be still unrecognized. Thus the concept can be expanded to potentially analyse the whole data set. However, this current report indicates the project was successful in confirming the hypothesis that mRNAs associated with proliferation, cytokine activity and adhesion can be observed to be regulated to develop a fertile milieu. We looked at the expression pattern of selected markers that had consistent changes, between LH+7 and LH+2 across women, overall pooling each cell type and marker, incorporating all samples demonstrated the stability of the development of the implantation process. We therefore established a pattern of expression common to all the women in the study. Prior to this work, consistency of markers expressed has been reported but consensus of which markers were involved in the process of the endometrium preparing a receptive WOI has not been achieved. This lack of knowledge has hindered progress in this field. Also, interestingly, not all markers were regulated similarly to all other markers, indicating the selectivity of the signalling pathways. Further, for some markers there was a clear distinction overall between regulation in stroma and glands, in particular for SMAD3, LIF, PCDH1, TRO, ITGB1 and ITGB3. This finding points to the importance of using defined cell types producing high quality mRNA and the value of the tissue bank generated for these studies. Our observations provide different information from others using multicellular tissue. We also performed analysis on subsets of the data. Comparison of changes of mRNA expression in separate cycles and in the same cycle compared to the overall pooled results of each cell type and marker showed there were few differences. It has been suggested that early wounding may result in subsequent increase of cytokine expression (Nastri et al., 2015). This finding was supported by others (Castelbaum et al., 1994; Creus et al., 2002; Ordi et al., 2002, 2003). We observed that sampling at LH+2 and LH+7 in the same cycle significantly increased only the mRNA expression of cytokines IL11 (glands) and CCL4 (stroma) and reduced expression of IL15 in glands at LH+7. This inability to confirm others could indicate that the increase of mRNA expression occurred in cell types other than the glands and stroma specifically studied here. Interestingly, a more recent Cochrane meta-analysis report reported findings that were more uncertain whether endometrial injury improved the success rates of pregnancy (Lensen et al., 2016). Further, we noted an absence of any change of pattern of expression of some cytokines, LIF, CX3CL1 and CCL19, when an increase had been reported by others (Dimitriadis et al., 2010). This finding was possibly a result of statistics being applied to this small population, or that those markers are either less likely to be involved in creating a WOI, or an adequate baseline expression of these mRNAs may be all that is required to develop a functionally receptive endometrium. On the other hand, in the adhesion molecule subset, ITGB1 and ITGB3, reported to be increased with wounding (Gnainsky et al., 2015), exhibited downregulation at LH+7 when two samples were taken in the same cycle. Further, numerous differences were detected for other adhesion markers in stroma when sampling twice in the same cycle. It is possible that this fragility of one segment of the adhesion control process hints at a potential clinical target for treating challenging infertility whereby the adhesion pathway may be excessively sensitive to an altered physiological environment. This study may indicate a general resistance to iatrogenic initiation due to the initial sampling at LH+2. Disadvantages in prior wounding in the same cycle as implantation have been reported (Nastri et al., 2015), because although cytokine activity may be increased, adhesion activity may be decreased. However, it is difficult to factor in pathway redundancies. Although we demonstrated that some mRNAs were altered by prior wounding, not all were sensitive and thus if they have a similar function and there is redundancy the non-sensitive mRNAs may be able to compensate. The constancy of the fertile ovulatory cycle is demonstrated in this report in endometrial mRNA expression by the limited expression differences between LH+2 when compared to LH+2 and LH+7 when compared to LH+7. Variation in endocrine factors related to endometrial receptivity have been reported (Santoro et al., 2000; Stricker et al., 2006; Mihm et al., 2011; Haggstrom, 2014) which could affect the cyclicity of the functionalis layer of the endometrium. Previous studies of fertile women involving a single endometrial sample but without repeat analyses in subsequent cycles (Ruiz-Alonso et al., 2012) did not address the question of whether a single assessment is valid for prediction of a subsequently functional WOI. Clinically, the repeated stable patterns over cycles suggest that a well-timed sample set of LH+2 and LH+7 from previous individual cycles of a woman will indicate if the woman will have a similar satisfactory endometrium in a subsequent cycle due to the apparently limited variation between cycles. The clear difference of expression between LH+2 and LH+7 indicates that any sampling must be rigorously timed. This finding has important clinical implications particularly about the possible effect of endometrial biopsies taken prior to IVF treatment (Narvekar et al., 2010; Nastri et al., 2015). In this study we undertook comparison between subsequent cycles of previously validated proliferative, cytokine and adhesion markers that had been justified by the literature. This enabled intracycle regulation of these markers to be identified for all participants as well as assessment of cycle variability. If a lag phase or alteration of the WOI is to be considered as a cause of unsuccessful implantation then confirmation of cycle to cycle consistency of IVF participants is required. Identification of an adequate number of mRNAs that are apparently regulated between the pre-receptive and the receptive phase appears to portray endometrial receptivity capability. Therefore, it is possible that a single mRNA or a small group of mRNAs may not provide sufficient information. A redundancy of mRNAs that can control the implantation process is likely and so that ultimately a panel of mRNAs will be required for development of a clinically applicable test. This proof of principle project gives us a better understanding of endometrial receptivity and may encourage the use of medical intervention or altered management of frozen embryo transfer that would be based upon an improved understanding of the natural fertile cycle. In summary, this paper has characterized specific cell types of endometrial stroma and glands. We investigated the behaviour of mRNAs that other workers had previously identified as being potentially involved in receptivity. Expression prior to and at the time of blastocyst’s implantation was determined in natural cycles, unaffected by exogenous drugs. We found the level of expression is robustly regulated and thus it is reasonable to infer that a woman who exhibits a fertile endometrial profile in one cycle will usually do so in subsequent cycles. This manuscript contributes to the fuller understanding of one of the multifactorial processes within the endometrium. Supplementary data Supplementary data are available at Human Reproduction online. Authors’ roles Gloria E. Evans—designed project, assisted with sample collection, performed laser microdissection and mRNA extraction, involved with data analysis and is the principal author of this paper. Gregory T.M. Phillipson—designed project, recruited volunteers, sampled women and assisted with the manuscript. Peter H. Sykes—assisted with project design, engaged in progress discussions throughout this study and assisted with the manuscript. Les A. McNoe—assisted preparing samples for microarrays, performed microarrays, data analysis and assisted with the manuscript. Cristin G. Print—assisted with design, performed the data analysis and assisted with the manuscript. John J. Evans—oversaw the complete project, assisted with project design, laboratory protocols, data analysis and assisted with the manuscript. Funding Funding was granted from the Tertiary Education Commission of New Zealand, Contract I.D.:UOOX06007. Conflict of interest None declared. Acknowledgements We wish to thank the wonderful women who volunteered for this study and to Fertility Associates, Christchurch, New Zealand for collection of these samples. 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Human Reproduction – Oxford University Press
Published: Mar 1, 2018
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