biology Article Semen Modulates Cell Proliferation and Differentiation-Related Transcripts in the Pig Peri-Ovulatory Endometrium Jaume Gardela 1,2,† , Mateo Ruiz-Conca 1,2,† , Dominic Wright 3 , Manel López-Béjar 2,4 , Cristina A. Martínez 1 , Heriberto Rodríguez-Martínez 1, *,‡ and Manuel Álvarez-Rodríguez 1,2,‡ 1 Department of Biomedical & Clinical Sciences (BKV), BKH/Obstetrics & Gynecology, Faculty of Medicine and Health Sciences, Linköping University, 58185 Linköping, Sweden; jaume.gardela@uab.cat (J.G.); mateo.ruiz@uab.cat (M.R.-C.); cristina.martinez-serrano@liu.se (C.A.M.); manualvro@gmail.com (M.Á.-R.) 2 Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; manel.lopez.bejar@uab.cat 3 Department of Physics, Chemistry and Biology, Faculty of Science and Engineering, Linköping University, 58185 Linköping, Sweden; dominic.wright@liu.se 4 College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766, USA * Correspondence: heriberto.rodriguez-martinez@liu.se † These authors contributed equally to this work. ‡ These authors contributed equally to this work. Simple Summary: Homeostasis of the uterus after mating is crucial for the subsequent reproductive events. The post-mating inflammatory response is restricted to the uterus, but semen also modulates the expression of other genes involved in regulation along the female reproductive tract, including the oviduct. This study aims to determine if several ejaculate fractions of the pig may modulate  cell proliferation and differentiation-related transcripts in different sections of the peri-ovulatory  sow reproductive tract. Our data demonstrate that most of the mRNA expression changes of the Citation: Gardela, J.; Ruiz-Conca, M.; 144 transcripts tested were induced by mating. Additionally, spermatozoa and seminal plasma also Wright, D.; López-Béjar, M.; Martínez, triggered differential expression of the transcripts tested. Finally, our data imply that spermatozoa, C.A.; Rodríguez-Martínez, H.; seminal plasma components, and the act of mating induce differential mechanisms in the peri- Álvarez-Rodríguez, M. Semen ovulatory female reproductive tract, which are essential for tissue repair. Modulates Cell Proliferation and Differentiation-Related Transcripts in Abstract: Uterine homeostasis is maintained after mating by eliminating pathogens, foreign cells, the Pig Peri-Ovulatory Endometrium. and proteins by a transient inflammation of the uterus. Such inflammation does not occur in the Biology 2022, 11, 616. https:// doi.org/10.3390/biology11040616 oviductal sperm reservoir (utero-tubal junction, UTJ), colonized by a population of potentially fertile spermatozoa before the inflammatory changes are triggered. Semen entry (spermatozoa Academic Editor: Paul S. Cooke and/or seminal plasma) modifies the expression of regulatory genes, including cell proliferation Received: 11 March 2022 and differentiation-related transcripts. Considering pigs display a fractionated ejaculation, this Accepted: 16 April 2022 study aims to determine whether different ejaculate fractions differentially modulate cell prolifera- Published: 18 April 2022 tion and differentiation-related transcripts in the sow reproductive tract during the peri-ovulatory stage. Using species-specific microarray analyses, the differential expression of 144 cell proliferation Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in and differentiation-related transcripts was studied in specific segments: cervix (Cvx), distal and published maps and institutional affil- proximal uterus (DistUt, ProxUt), UTJ, isthmus (Isth), ampulla (Amp), and infundibulum (Inf) of iations. the peri-ovulatory sow reproductive tract in response to semen and/or seminal plasma cervical deposition. Most mRNA expression changes were induced by mating. In addition, while mating upregulates the fibroblast growth factor 1 (FGF1, p-value DistUt = 0.0007; ProxUt = 0.0253) tran- script in the endometrium, both its receptor, the fibroblast growth factor receptor 1 (FGFR1, p-value Copyright: © 2022 by the authors. DistUt = 2.14 e−06 ; ProxUt = 0.0027; UTJ = 0.0458) transcript, and a potentiator of its biological effect, Licensee MDPI, Basel, Switzerland. the fibroblast growth factor binding protein 1 (FGFBP1), were downregulated in the endometrium This article is an open access article (p-value DistUt = 0.0068; ProxUt = 0.0011) and the UTJ (p-value UTJ = 0.0191). The FGFBP1 was distributed under the terms and downregulated in the whole oviduct after seminal depositions (p-value Isth = 0.0007; Amp = 0.0007; conditions of the Creative Commons Attribution (CC BY) license (https:// Inf = 6.87 e−05 ) and, interestingly, FGFR1 was downregulated in the endometrium in the absence of se- creativecommons.org/licenses/by/ men (p-value DistUt = 0.0097; ProxUt = 0.0456). In conclusion, the findings suggest that spermatozoa, 4.0/). Biology 2022, 11, 616. https://doi.org/10.3390/biology11040616 https://www.mdpi.com/journal/biology Biology 2022, 11, 616 2 of 16 seminal components, and the act of mating trigger, besides inflammation, differential mechanisms in the peri-ovulatory female reproductive tract, relevant for tissue repair. Keywords: cell proliferation; cell differentiation; inflammation; interleukins; chemokines; fibroblast growth factors; insulin-like growth factors; pig 1. Introduction Infertility is a complex component of reproductive health [1]. With the limitations of bioethics on human research, animal models have become a crucial tool for developing such studies [2]. The porcine model presents several similarities with humans, including genetics, anatomy, and physiology [3]. Further research is needed to highlight the importance of the subjacent roles of the molecules involved in infertility in both human and animal models, such as the pig, for the study of reproduction [3,4]. Fertility depends upon a correct balance between pro-and anti-inflammatory compo- nents, warranting homeostasis [5,6]. Inflammation is a part of relevant steps of reproductive physiology, present during ovulation, mating, sperm tolerance, placentation, or parturi- tion [7]. The role of semen and seminal plasma (SP) triggering temporary inflammation in the female reproductive tract has been demonstrated in most species [8], including human [9], mice [10], bovine [11], equine [12], porcine [13], and lagomorpha [14]. This tem- poral post-mating inflammation removes potential pathogens and exogenous SP-proteins, as well as the spermatozoa that do not reach the utero-tubal junction [15]. The endometrium shows essential, hormonally driven, plastic changes along the estrous cycle and embryo implantation [16], primarily influenced by estrogen and progesterone [17], but also influ- enced by conceptus-derived signals [18]. These endometrial changes involve apoptotic and cell proliferation and differentiation processes [19]. However, whether the semen components (spermatozoa and/or SP) influence expression changes of cell proliferation and differentiation transcripts in the peri-ovulatory reproductive tract is still unknown. The inflammasomes are large multiprotein oligomers known for contributing to the sensing of pathogen- and damaged-associated molecular patterns triggered by a wide range of substances that emerged during infections, tissue damage, or metabolic imbal- ance [20,21]. For instance, the nucleotide-binding leucine-rich repeat receptor 3 (NLRP3), a sensor molecule for the formation of inflammasomes, has been pointed as critically involved in the induction of the inflammation [22–24], and responsible, at least in part, of preeclampsia in humans when upregulated [25]. The NLRP3-inflammasome interacts with well-characterized inflammation and immune reaction pathways, such as insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs). The IGFs are single-chain polypeptides, structurally related to insulin, playing vital roles in different key processes, including the growth and proliferation of embryos, tissue differentiation, cell survival, and proliferation [26]. The biological effects of IGFs are mediated by a family of cell-surface receptors that includes insulin receptors and IGF receptors (IGFRs) [27]. Additionally, the biological actions of IGFs are modulated by the IGF binding proteins (IGFBPs) [27,28]. The IGF system is essential for preimplantation and placenta development [29]. Local production of IGFs has been reported in the uterus and placenta of several species [30–32], including the pig [33–35]. The FGFs integrate a large family of polypeptides growth fac- tors with a large variety of functions in development, wound repair, angiogenesis, and metabolism [36,37]. They are sequentially named (FGF1-23) requiring heparan sulfate to activate one of four cell-surface FGF receptors [36]. The FGF family is a multifunctional regulator of differentiation, migration, and cell growth processes [36,37]. The overall aim is to study changes in cell proliferation and differentiation-related transcripts in the peri-ovulatory sow reproductive tract in response to natural mating (i), the insemination with either the first portion of the sperm rich fraction of the ejaculate (ii), the cervical deposition of the SP of the first fraction (iii), or the SP of the complete ejaculate (iv). Biology 2022, 11, 616 3 of 16 The hypothesis tested is that semen components (spermatozoa and/or SP) act differentially on specific segments of the genital tract, comparing the tubal sperm reservoir, where fertile spermatozoa escape the post-mating inflammatory response, whereas in the endometrium, the inflammatory cleansing requires tissue repair prior to implantation. 2. Materials and Methods 2.1. Ethics Permit Animal handling and experiments were carried out according to the European Com- munity Directive 2010/63/EU, 22 September 2010, and Swedish legislation (SJVFS 2017:40). The study was accepted by the Regional Committee for Ethical Approval of Animal Experi- ments (Linköpings Djurförsöksetiska nämnd, Linköping, Sweden). Permits number 75-12 (10 February 2012), ID1400 (2 February 2018), and Dnr 03416-2020 (26 March 2020). 2.2. Animal Handling and Tissue Collection Weaned sows (parity 1–3, n = 20) and matured boars (9–11 months of age, n = 5) of Swedish Landrace breed (Sus scrofa domestica) were held under temperature and light control in separate pens at the Translational Medicine Centre (TMC/CBR-3) of Linköping University. Animals were fed with commercial feedstuff, and water was provided ad libitum. Sows were exposed to snout-to-snout contact with adjacent boars while pro-estrus and estrus behavior signs were checked by experienced staff, applying back pressure to sows two times per day. When sows presented estrus reflex (first day of behavioral estrus), they were randomly assigned to different experimental groups. Control females were cervically infused with protein-free Beltsville thawing solution (Control group, n = 4), while experimental females were cervically infused with pooled sperm rich-fraction of the ejaculate (Semen-AI group, n = 4), pools of sperm-free SP of the whole ejaculate (SP-Total group, n = 4), sperm-free SP harvested from pooled sperm-peak fractions (SP-AI group, n = 4), or mated with a single male per sow, using 4 different males in total (Natural Mating group, n = 4) as previously described [38,39]. After 24 h of each treatment, sows were subjected to general anesthesia. The mucosa of the tubular reproductive tract (right side) was exposed and collected at specific segments: cervix (Cvx), distal uterus (DistUt), proximal uterus (ProxUt), utero-tubal junction (UTJ), isthmus (Isth), ampulla (Amp), and infundibulum (Inf). Mucosal samples were directly plunged into liquid nitrogen and stored in cryovials at −80 ◦ C for mRNA expression analyses. 2.3. Collection of Semen and Seminal Plasma Semen and SP were collected following prior methods [38,39]. The gloved-hand approach was used to collect semen once a week. Sperm concentration and motility were assessed in all samples (QualiSperm; AKYmed, Cheseaux-sur-Lausanne, Switzerland). Ejaculates used for the study had more than 70% motility and 75% spermatozoa with normal morphology. After double centrifugation at 1500× g for 10 min and checking for the absence of sperm cells, SP was collected from the total ejaculate or the sperm-rich fraction. 2.4. Transcriptome Analysis and Bioinformatics Total RNA from reproductive samples was extracted following a TRIzol modified protocol [38,39]. RNA concentration, integrity evaluation, cDNA synthesis, and microarray analyses (GeneChip® Porcine Gene 1.0 ST Array, Affymetrix Inc., 3420 Central Expressway, Santa Clara, CA, USA) were performed according to methods previously described [38,39]. Only samples with RNA integrity values larger than 9 were employed for microarray hybridization. The GeneChip® Whole Transcript Plus reagent kit (Affymetrix, Santa Clara, CA, USA) was used to synthesize cDNA (250 ng/reaction). An initial incubation of the hybridization cocktail at 99 ◦ C for 5 min was done after a fall to 45 ◦ C before loading the microarray (GeneChip® Porcine Gene 1.0 ST Array, Affymetrix Inc., 3420 Central Expressway, Santa Clara, CA, USA). The cocktail hybridization solution (130 µL) was then put into each microarray and incubated for 16 h at 45 ◦ C under 60 rotations/min. The Santa Clara, CA, USA) was used to synthesize cDNA (250 ng/reaction). An initial incuba- tion of the hybridization cocktail at 99 °C for 5 min was done after a fall to 45 °C before loading the microarray (GeneChip® Porcine Gene 1.0 ST Array, Affymetrix Inc., 3420 Cen- tral Expressway, Santa Clara, CA, USA). The cocktail hybridization solution (130 μL) was Biology 2022, 11, 616 4 of 16 then put into each microarray and incubated for 16 h at 45 °C under 60 rotations/min. The hybridized microarray chip was unloaded after incubation and washed and stained with the GeneChip® Fluidics Station 450 (Affymetrix, Santa Clara, CA, USA) before being hybridized scanned with microarray chip wasGeneChip the Affymetrix unloaded® after incubation Scanner GCS3000 and(Affymetrix, washed andSanta stained with CA, Clara, the GeneChip ® Fluidics Station 450 (Affymetrix, Santa Clara, CA, USA) before being scanned USA). with the Affymetrix GeneChip ® Scanner GCS3000 (Affymetrix, Santa Clara, CA, USA). Data processing was previously described [38,39], by using the Transcriptome Anal- Data processing was previously described ysis Console (TAC; version 4.0) from Affymetrix. [38,39], by using Briefly, thethe Transcriptome array Analysis chip data were pro- Console (TAC; version 4.0) from Affymetrix. Briefly, the array chip cessed using robust multi-array average (RMA) normalization, computing average inten-data were processed using robust by sity values multi-array background average (RMA) normalization, adjustment, computingamong quantile normalization averagemicroarrays, intensity values and by background finally, adjustment, quantile log2 transformation normalization for extracting amongvalues the expression microarrays, of each and finally,in transcript log2 the transformation probe set. The for extracting normalized the expression mRNA expressionvalues data ofof the each144transcript selected in the probewere transcripts set. The normalized analyzed, mRNA expression for validation, using thedata of the 144Rselected open-source transcripts were [40]. Differentially mRNA analyzed, for expression validation, using the open-source R [40]. Differentially mRNA expression was calculated using a linear model and the empirical Bayes’ approach implemented in was calculated using a linearlimma the package model and [41]. the empirical Control Bayes’ type-I errors approach were checkedimplemented in the package using a Benjamini–Hochberg limma [41]. Control type-I errors were checked using a Benjamini–Hochberg false discovery rate (q < 0.05) and a principal component analysis-based p-value falsecorrection. discovery rate (q < 0.05) and a principal component analysis-based p-value correction. Gene Ontology (GO) terms and pathways were analyzed by the PANTHER classification Gene Ontology (GO) systemterms and pathways (protein annotation were analyzed through by the PANTHER evolutionary classification relationship) system [42] based (protein on the Kyoto annotation Encyclopedia of Genes and Genomes database (KEGG) [43]. Graphical illustration of through evolutionary relationship) [42] based on the Kyoto Encyclopedia of Genes and Genomes overrepresented database GO terms and (KEGG) pathways [43]. wasGraphical produced withillustration of overrepresented the Cytoscape v3.0.0 appli- GO terms and pathways was produced with the Cytoscape v3.0.0 application ClueGO cation ClueGO v2.0.3 [44] and principal component analysis (PCA) and heatmaps were v2.0.3 [44] and principal component analysis (PCA) and heatmaps were performed by performed by using the ClustVis web tool [45]. using the ClustVis web tool [45]. 3.3.Results Results ThePCA The PCAanalysis analysisof ofthe the144 144 transcripts transcripts depicted depicted an an appropriate appropriate division division of of the the treat- treat- ment groups (Figures 1 and S1) and the grouping factor by heatmaps of the chosen ment groups (Figure 1 and Figure S1) and the grouping factor by heatmaps of the chosen genes was conserved genes acrossacross was conserved tissues analyzed tissues (Figure analyzed S2). S2). (Figure Figure 1. Figure 1. Principal Principalcomponent component analysis of the analysis genes of the included genes in this included instudy of natural this study mating,mating, of natural Semen- Semen-AI, SP-AI; and SP-Total groups. (a). DistUt: distal uterus; (b). ProxUt: proximal uterus;UTJ: AI, SP-AI; and SP-Total groups. (a). DistUt: distal uterus; (b). ProxUt: proximal uterus; (c). (c). utero-tubal UTJ: junction. utero-tubal junction. Theofficial The officialnames namesofofthe thegenes genesand andthe theSus Susscrofa scrofaannotation annotationininthe theKEGG KEGGpathways pathways database [43] was used to find the differentially expressed genes with different biological database [43] was used to find the differentially expressed genes with different biological pathwaysand pathways andGOGObiological biologicalprocess processsubgroups subgroups(Figure (Figure2). 2). The differential expression of the 144 selected transcripts showed the changes were in response to the natural mating, mostly downregulated (Figure 3a and Table S1). Moreover, some transcriptomic changes were found in the groups containing the first portion of the sperm-rich fraction (Semen-AI; Figure 4a) and only SP, either from the first fraction of the ejaculate (SP-AI; Figure 5a) or from the entire ejaculate (SP-Total; Figure 6a). Biology 2022, x FOR PEER REVIEW 2022, 11, 616 5 5of of 16 Figure 2. Schematic representation of altered transcripts of interest among all tissues and treatments. The Figure 2. Schematic of over-representation representation of altered functional categories was transcripts of using carried out interest theamong all tissues Cytoscape v3.0.0and treatments. (ClueGo v2.0.3). The over-representation of functional categories was carried out using the Cytoscape Gene Ontology (GO) subgroups biological process are depicted as circles. Terms are grouped v3.0.0 (ClueGo based v2.0.3). Gene Ontology (GO) subgroups biological process are depicted as circles. Terms are on common genes function (kappa score) and discriminated by colors. The size of the nodes shows grouped based on common genes function (kappa score) and discriminated by colors. The size of the degree of significance (low to high significance, respectively). ClueGo parameters of biological the nodes shows the degree of significance (low to high significance, respectively). ClueGo param- process; GO tree levels, eters of biological 2–4 GO process; (from 0);levels, tree minimum number 2–4 (from 0);of genes, 1; number minimum minimum of percentage of genes, genes, 1; minimum percentage of genes, 1.2; GO term fusion; GO term connection restriction (kappa score), 0.4; GOinitial 1.2; GO term fusion; GO term connection restriction (kappa score), 0.4; GO term grouping, term group sizeinitial grouping, of 2 and 50%size group for group merge. of 2 and 50% for group merge. The differential expression of the 144 selected transcripts showed the changes were in response to the natural mating, mostly downregulated (Figure 3a and Table S1). More- over, some transcriptomic changes were found in the groups containing the first portion of the sperm-rich fraction (Semen-AI; Figure 4a) and only SP, either from the first fraction of the ejaculate (SP-AI; Figure 5a) or from the entire ejaculate (SP-Total; Figure 6a). Biology 2022, 11, x FOR PEER REVIEW 6 of 16 Biology 2022, 11, 616 6 of 16 Figure 3. Summary of the transcriptomic analysis in the natural mating group. (a) Volcano plot Figure 3. Summary of the transcriptomic analysis in the natural mating group. (a) Volcano plot rep- representation of differential expression analysis of transcripts in the natural mating versus negative resentation of differential expression analysis of transcripts in the natural mating versus negative control comparison. Red dots represent upregulated transcripts and green dots downregulated control comparison. Red dots represent upregulated transcripts and green dots downregulated transcripts.Black transcripts. Blackdots dotsrepresent representall alltranscripts transcriptsthat thatshowed showedan analtered alteredexpression expressionprofile. profile.The Thex-axis x-axis showsthe shows thelog2 log2fold-changes fold-changesininexpression expressionand andthe they-axis y-axisthe thestatistical statisticalsignificance (−log10p-value). significance(−log10 p-value). (b) Box-plot (b) Box-plotrepresentation representationof ofthe thelog2 log2signal signalintensity intensityofof the the selected selectedtranscripts transcriptsby bytissue: tissue:Cvx: Cvx: cervix; cervix;DistUt: DistUt:distal distaluterus; uterus;ProxUt: ProxUt:proximal proximaluterus; uterus;UTJ: UTJ:utero-tubal utero-tubaljunction; junction;Isth: Isth:isthmus; isthmus;Amp: Amp: ampulla; ampulla;and andInf: Inf:infundibulum. infundibulum.* *pp<<0.05 0.05relative relativetotonegative negativecontrol. control. Biology 2022, 11, x FOR PEER REVIEW 7 of 16 Biology 2022, 11, 616 7 of 16 Figure 4. Summary of the transcriptomic analysis in the Semen-AI group. (a) Volcano plot repre- Figure 4. Summary of the transcriptomic analysis in the Semen-AI group. (a) Volcano plot repre- sentation of differential expression analysis of transcripts in the Semen-AI versus negative control sentation of differential expression analysis of transcripts in the Semen-AI versus negative control comparison.Red comparison. Reddots dotsrepresent representupregulated upregulatedtranscripts transcriptsand andgreen greendots dotsdownregulated downregulatedtranscripts. transcripts. Black dots represent all transcripts that showed an altered expression profile. The Black dots represent all transcripts that showed an altered expression profile. The x-axis shows x-axis showsthe the log2 fold-changes in expression and the y-axis the statistical significance ( − log10 p-value). log2 fold-changes in expression and the y-axis the statistical significance (−log10 p-value). (b) Box- (b) Box- plotrepresentation plot representation of the log2 signal signal intensity intensityof ofthe theselected selectedtranscripts transcriptsbybytissue: tissue:Cvx: Cvx:cervix; DistUt: cervix; Dis- tUt: distal uterus; ProxUt: proximal uterus; UTJ: utero-tubal junction; Isth: isthmus; Amp: distal uterus; ProxUt: proximal uterus; UTJ: utero-tubal junction; Isth: isthmus; Amp: ampulla; and ampulla; and Inf: Inf: infundibulum. infundibulum. *p< * p0.05 < 0.05 relative relative to negative to negative control. control. Biology 2022, 11, x FOR PEER REVIEW 8 of 16 Biology 2022, 11, 616 8 of 16 Figure 5. Summary of the transcriptomic analysis in the SP-AI group. (a) Volcano plot representation Figure 5. Summary of the transcriptomic analysis in the SP-AI group. (a) Volcano plot representa- of differential expression analysis of transcripts in the SP-AI versus negative control comparison. tion of differential expression analysis of transcripts in the SP-AI versus negative control compari- Green dots represent downregulated transcripts. Black dots represent all transcripts that showed son. Green dots represent downregulated transcripts. Black dots represent all transcripts that an altered showed expression an altered profile.profile. expression The x-axis The shows the log2 x-axis shows thefold-changes in expression log2 fold-changes and the in expression and y-axis the the statistical y-axis significance the statistical (−log10 significance p-value). (−log10 (b) Box-plot p-value). representation (b) Box-plot of theoflog2 representation signal the log2 intensity signal in- of the of tensity selected transcripts the selected by tissue: transcripts Cvx: cervix; by tissue: DistUt:DistUt: Cvx: cervix; distal uterus; ProxUt: distal uterus; proximal ProxUt: uterus; proximal uterus; UTJ: utero-tubal UTJ: utero-tubal junction; junction; Isth: isthmus; Isth: isthmus; Amp: ampulla; Amp: ampulla; and Inf: and Inf: infundibulum. infundibulum. * p < 0.05*relative p < 0.05to relative negativeto control. negative control. Biology 2022, 11, x FOR PEER REVIEW 9 of 16 Biology 2022, 11, 616 9 of 16 Figure 6. Summary of the transcriptomic analysis in the SP-Total group. (a) Volcano plot repre- Figure 6. Summary of the transcriptomic analysis in the SP-Total group. (a) Volcano plot represen- sentation of differential expression analysis of transcripts in the SP-Total versus negative control tation of differential expression analysis of transcripts in the SP-Total versus negative control com- comparison. Red dots represent upregulated transcripts and green dots downregulated transcripts. parison. Red dots represent upregulated transcripts and green dots downregulated transcripts. Blackdots Black dotsrepresent representall alltranscripts transcriptsthat thatshowed showedan analtered altered expression expression profile. profile. The The x-axis x-axis shows shows the the log2 fold-changes in expression and the y-axis the statistical significance ( − log10 p-value). log2 fold-changes in expression and the y-axis the statistical significance (−log10 p-value). (b) (b) Box- Box- plotrepresentation plot representationofofthe the log2 log2 signal intensity intensityof ofthe theselected selectedtranscripts byby transcripts tissue: Cvx: tissue: cervix; Cvx: DistUt: cervix; Dis- distal uterus; ProxUt: proximal uterus; UTJ: utero-tubal junction; Isth: isthmus; Amp: tUt: distal uterus; ProxUt: proximal uterus; UTJ: utero-tubal junction; Isth: isthmus; Amp: ampulla;ampulla; and and Inf:Inf: infundibulum. infundibulum. * p *