Endometriosis is a common gynecological disease with high prevalence, while its etiology and pathophysiology have remained to be fully elucidated. Previous evidence suggested that this disorder may be in part or completely of somatic origin. However, traditional endometrial samples may not be ideal for investigation, as target cells, including epithelial and stromal cells, in endometriotic lesions are too sparse to be analyzed. Recently, capture microdissection techniques have been used to overcome these limitations and eliminate tissue heterogeneity in endometriosis research. Therefore, the present review summarized the alterations in epithelial and stromal cells in endometriosis tissues isolated through capture microdissection, outlined recent progress and provided directions for future investigation of the pathogenesis of endometriosis.
Endometriosis is a chronic and inflammatory disorder caused by the presence of endometrial tissues, including endometrial glandular and stromal cells, outside the uterine cavity. As one of the most common gynecological diseases, it affects 6–10 % of women of reproductive age (
The pathogenesis of endometriosis is generally explained using Sampson's retrograde menstruation theory. This theory suggests that endometrial cells are implanted into the peritoneum or pelvic organs through reflux via fallopian tubes during menstruation and is supported by several lines of evidence. Epidemiological studies showed that a long menstrual duration (menstrual flow at least 6 days per month) and short menstruation periods (less than 27 days) are risk factors for endometriosis (
The occurrence of endometriosis in only a small proportion of women who had retrograde menstruation may be due to unique characteristics of their endometrial fragments, which have a key role in the pathogenesis of this disease. Thus far, intrinsic differences have been detected among ectopic and eutopic endometrium in endometriosis, and normal endometrium in controls (
This review summarizes recent advances in endometriosis research using capture microdissection approaches and discusses how these techniques can be improved and utilized in future studies.
Clonality analysis is used to study the development of tumors and malignancies and identify their neoplastic characteristics (
Additionally, it is worth mentioning that whether genomic alterations occur in the functional and/or basal layer of the endometrium prior to menstrual reflux remains ambiguous. The functional layer of the endometrium can regenerate at each menstrual cycle and can thus easily disrupt the genomic integrity of the endometrium. However, increasingly sophisticated clonality analysis has been associated with the proposal of the ‘stem cell theory of endometriosis’. Based on the regenerative physiological characteristics of endometrium, it has been suggested that endometrial stem cells may be derived from the basal layer of the uterus (
LOH refers to a gross chromosomal event that leads to loss of the entire gene and the surrounding chromosomal region (
Furthermore, the locus associated with TP53 is among the most commonly investigated gene locations; However, thus far, TP53 mutations have only been found in endometriosis specimens coexisting with carcinoma but not in solitary endometriotic lesions (
In summary, the highest LOH frequencies were detected on chromosome 10q23.3 and the second-highest on chromosome 9p21. Results on other LOH events were inconsistent among the studies included in the present review and therefore require clarification by additional investigations. Although these data cannot fully support the hypothesis that endometriosis is a true neoplasm, they may imply that downregulation of certain TSGs in endometriosis is one mechanisms of endometriosis pathogenesis and is therefore worthy of further in-depth study.
CNVs are thought to account for ~1% of the variation between two individuals and single nucleotide polymorphisms (SNPs) are thought to account for ~0.1% (
To date, despite the various chromosomal alterations detected, few of these changes have been observed by more than one study. Of particular concern is that no specific
At the transcript level, gene expression in ectopic and eutopic endometria from patients with endometriosis significantly differs from that in healthy women (
Matsuzaki
Microarray data revealed that three candidate genes, namely tyrosine kinase receptor B (TrKB), μ-opioid receptor (MOR) and serotonin transporter, are potentially involved in pain pathways of endometriosis (
The two aromatase expression regulators chicken ovalbumin upstream promoter transcription factor 2 (COUP-TF2) and prostaglandin E2 receptor subtype EP3 (PGE2EP3) were evaluated by microarray analyses (
Considering molecular defects in the endometrium involved in implantation and endometriosis-associated infertility, Matsuzaki
Three DNA methyltransferases (DNMT), namely DNMT1, DNMT3A and DNMT3B, cooperatively maintain DNA methylation and are overexpressed in ectopic endometrium compared with that in normal control subjects or eutopic endometrium from women with endometriosis (
While the scope of gene function research is broad, only 28 DEGs have been identified in endometriosis research using microdissection techniques. Research has not only focused on elucidating the underlying molecular mechanisms of endometriosis but also on its clinical symptoms, such as pain and infertility. Analysis of specific molecular markers is one potential option for non-invasive diagnosis of endometriosis, which may also be utilized as molecular targets for its selective and efficient treatment and prevention.
Several lines of evidence have indicated that genomic alterations in the endometrium are essential for the development of endometriosis. However, the precise molecular mechanisms underlying the pathogenesis of endometriosis remain elusive. Based on the current progress in endometriosis research, two interesting topics are discussed here. Clear interpretation of these issues can provide experiences to other investigators and anticipate future directions in endometriosis research.
Hypothesis-based versus hypothesis-free research. Studies of endometriosis-associated genes are classified into hypothesis-based and hypothesis-free approaches (
In the present review, three important methods, namely CGH, SNP array and whole exome sequencing (WES), which were used in previous studies to detect alterations associated with endometriosis without prior knowledge of specific regions of interest, were compared. The use of the traditional CGH method has detected numerous cryptic gains and/or losses of chromosomal regions in endometriosis samples. However, the application of CGH has been hampered by limited resolution and low throughput. The SNP array and WES can offer accurate and high-throughput detection. However, in studies by Saare
The capture microdissection technology was first described in the early 20th century and has been progressively optimized in recent years. During MCM, defined cell populations are manually cut out from tissue sections under the microscope. Although this method is feasible for isolation of specific cells, it proved to be inefficient and time consuming (
In the present review, advances in endometriosis research using capture microdissection techniques were presented. Evidence from 32 studies revealed various somatic alterations in ectopic and eutopic endometrosis tissues. These results enhance the current understanding of the pathogenesis of endometriosis and provide putative biomarkers as well as therapeutic targets. With the rapid advancement of detection technologies, future studies should take into account the use of the hypothesis-free method combined with microdissection techniques for precise analysis. For example, single-cell high-throughput sequencing combined with LCM can be used not only in genomics but also in transcriptomics and proteomics studies. Furthermore, hypothesis-based studies with an appropriate design and large sample size must be performed to confirm preliminary data from hypothesis-free studies. With the application of capture microdissection, great advances in the elucidation of the pathogenesis of endometriosis are expected in the near future.
The present study was supported by the National Natural Science Foundation of China (no. 81571411).
Results of clonality analyses of microdissected ectopic endometrial tissues from endometriosis patients.
Author, year | Samples/informative samples (n) | Monoclonal samples, n (%) | Assay | Refs. |
---|---|---|---|---|
Jiang, 1996 | 20/17 | 14 (82) | MCM/HUMARA | ( |
Tamura, 1998 | 25/10 | 10 (100) | MCM/PGK | (18) |
Wu, 2003 | 40/38 | 38 (100) | LCM/HUMARA | ( |
Nabeshima, 2003 | 22/9 (PGK) | 9 (100) | LCM/HUMARA, PGK | ( |
Nabeshima, 2003 | 22/18 (HUMARA) | 18 (100) | LCM/HUMARA, PGK | ( |
Mayr, 2003 | 29/13 | 2 ( |
LCM/PGK | ( |
Wang, 2010 | 50/34 | 34 (100) | LCM/HUMARA | ( |
MCM, manual capture microdissection; LCM, laser capture microdissection; HUMARA, human androgen receptor gene assay; PGK, phosphoglycerate kinase.
Results of loss of heterozygosity analysis of microdissected endometrium of patients with endometriosis.
Sample type | |||||
---|---|---|---|---|---|
Author, year | Cases (n) | Controls (n) | Analyzed chromosomes | Frequent changes: Patients with alterations (n)/patients, n (%) | Refs. |
Jiang, 1996 | EC:E+S ( |
Blood ( |
16q14–15(D6S284);6q14(D6S300); 9p21(D9S171); | 9p21(D9S171):3/34(8.8) | ( |
9p21(D9S161);1q23.3(D11S1336); | 9p21.2(D9S161):6/34(17.6) | ||||
11q23.3(D11S1328);17p13.1(TP53CA); | 11q23.3(D11S1336):5/33( |
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17q21.1(D17S855); | 11q23.3(D11S1328):1/33( |
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17q21.1(D17S806);22q13-qter(D22S304); | 22q13-qter(D22S304):2/40( |
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22q12.3–13.1(PDGFB);22q11.2-qter(CYP2D); | 22q11.2-qter(CYP2D):4/40( |
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22q12.1-ter(D22S276);22q13.3(D22S274); Xq11.1(HUMARA-1); | 22q12.1-ter(D22S276):2/40( |
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Sato, 2000 | EC:E ( |
Normal epithelial | 10q23.3(D10S215); | 10q23.3 (D10S215):3/11(27.3); | ( |
cells |
10q23.3(D10S608); | 10q23.3 (D10S608):6/12(50.0); | |||
10q23.3(D10S541); | 10q23.3 (D10S541):8/14(57.1); | ||||
Nakayama, 2001 | EC:E ( |
EU:E ( |
9p21(P16);9p22.3(PTCH);11p13(WT1);9p21.2(D9S161);13q14.2(RB1); | 9p22.3 (PTCH):1/4( |
( |
14q32(D14S267);17q21.3(NM23);18q21.1(DPC-4);18q21.3(DCC) | |||||
Goumenou, 2001 | EC:E ( |
EU:E ( |
1p31-p21(D1S116);1p(D1S186);1q42-q43(ACTN2); | 1q21q23 (APOA2):1/18(5.5); | ( |
1q21q23(APOA2);2p(D2S2291);2p(D2S288); | 9p21 (D9S161):3/20(15.0); | ||||
2q21-q33(D2S141);2q32(D2S202);2q(D2S311); | 9p21 (D9S270):3/17(17.6); | ||||
3p24.2-p22(D3S1561);p21(D9S161);9p21(D9S270); | 9p21 (D9S265):1/15(6.7); | ||||
9p22(IFNA);9p21(D9S265);9q31-q34(D9S112); | 17q11.2–12.1(D17S250):1/21(4.8) | ||||
17p13.1(TP53);17q11.2–12.1(D17S250); | |||||
Guo, 2004 | EC:E ( |
NU:E ( |
19q13.31(D19S408); 1p34.3–36.11(D1S3463) | 1p34.3-36.11(D1S3463):1/5( |
( |
epithelial cells ( |
6p22.3–24(D6S1653) | 6p22.3–24(D6S1653):1/5( |
|||
Prowse, 2005 | EC:E ( |
EC:S ( |
9p21.3(D9S1870);9p21.3(D9S265); | 8p22 (D8S261):1/14( |
( |
9p21.1(D9S270);9p21.2(D9S161);11q23.3(D11S29); | |||||
1p36.13(D1S199);8p22(D8S261);1q23.3(APOA2); | |||||
9q22.3(PTCH); 17p13.1(TP53); | |||||
10q23.31(D10S541);10q23.31(D10S1765) | |||||
Ali-Fehmi, 2006 | EC:E ( |
EC:S ( |
10q23.3(D10S215); | 10q23.3 (D10S215):1/23(4.3) | ( |
10q23.3(D10S608); | 10q23.3 (D10S608):2/23(8.7); | ||||
10q23.3(D10S541); | 10q23.3 (D10S541):4/23(17.4) | ||||
Xu, 2011 | EC:E ( |
EC:S ( |
5q11.2(D5S407);6p24.3(D6S470); | 9p21.1 (D9S270):1/12(8.3) | ( |
9p21.1(D9S270); | |||||
9p21.2(D9S161);9p21.3(D9S265);9p21.3(D9S1870); | 9p21.2 (D9S161):1/12(8.3) | ||||
10q23.31(D10S541);10q23.31(D10S1765); | 10q23.31(D10S541):3/12( |
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10q23–24(D10S215);10q23–24(D10S579); 13q31(D13S265); | 13q31(D13S265):3/12( |
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17p13.1(TP53);17q22–23.2(D17S787); | |||||
Wang, 2012 | EC:E (LCM:22; MCM:18) | Normal ovarian | 10q23.31(TTTA1);10q23.31(TTTA2); | 17q21.31(AAAT2); | ( |
epithelial cells | 9p21.3(AAGG);9p21.3(AAGA); | 9p21.3(AAAT1); | |||
(LCM:22; MCM:18) | 9p21.3(AAAT1);6p21.31(GATA61E03); | 10q23.31(TTTA1); | |||
6p21.31(TTTC1);6p21.31(TTTC2); | LCM: 13/22(56.09); | ||||
17p13.1(TCTT);17p13.1(D17S720); | MCM: 11/18(61.11); | ||||
17q21.31(CTTT);17q21.31(ACA); | |||||
17q21.31(AAAT2);22q12.3(TTCC); 22q12.3(TAA) | |||||
Saare, 2012 | EC:E ( |
EU:E + blood ( |
The whole genome | No foci-specific cn-LOH | ( |
Epithelial cells from fallopian tubes and lymph nodes. EC, ectopic endometrium; EU, eutopic endometrium; E, glandular epithelial cell; NU, normal endometrium; MCM, manual capture microdissection; LCM, laser capture microdissection; S, stromal cell; cn-LOH, copy neutral loss of heterozygosity.
Results of genome-wide copy number variation and single nucleotide variation analysis of microdissected endometrium of patients with endometriosis.
Author, year | Samples (n) | Patients with alterations (n/total) | Total CNVs/SNVs | Frequent changes | Methods | Refs. | |
---|---|---|---|---|---|---|---|
Gogusev, 1999 | EC ( |
15/18 | 59 | G: 6q, 7q, 17q; | MCM, CGH | ( |
|
L: 1p, 5p, 6q,7p14–22qter, | |||||||
22q12.3-qter, 9q, 16q, 17q | |||||||
Gogusev, 2000 | FbEM1 cell line | 1/1 | 66 | G: 1, 2, 3, 5, 6p, 7, | MCM, CGH | ( |
|
+ original | 16, 17, 20, 21q, 22q; | ||||||
endometriotic | L: 6q,9,11p, | ||||||
sample ( |
12,13q,18, X | ||||||
Guo, 2004 | EU ( |
5/5 | 68 | G: 3p, 10q, 3q; | LCM, aCGH | ( |
|
L: 1p, 3p, 4p, 22q | |||||||
Wu, 2006 | EC ( |
5/5 | 810 (EC) + 745 (EU) | G: 1p, 5p, 6p+q, 11p,Xq; | LCM, aCGH | ( |
|
L: 1p, 5p, 6q, 16q | |||||||
Saare, 2012 | EC ( |
0/11 | No |
LCM, aSNP | ( |
||
+ blood ( |
|||||||
Silveria, 2012 | EC ( |
8/8 | 119 | G: 1q21-q23, 11q12-q21; | LCM, | ( |
|
L: 3p24-p25, 5q34, 7p14-p21, | HR-CGH | ||||||
9p21, 11q23q24, 16q22-q23, | |||||||
18q12-q21, 18q22-q23, 9q13 | |||||||
Li, 2014 | EC ( |
16/16 | No overlapped | LCM, WES | ( |
||
NU ( |
SSNVs |
EU, eutopic endometrium; NU, normal endometrium; EC, ectopic enmometrium; CNV, copy number variation; SNV, single nucleotide variation; MCM, manual capture microdissection; LCM, laser capture microdissection; aCGH, array comparative genomic hybridization; WES, whole exome sequencing; G, gains; L, losses; SCNA, somatic copy number alteration; SSNV, overlapping single nucleotide variation; aSNP, array single nucleotide polymorphism.
Differentially expressed genes in microdissected endometrium of patients with endometriosis.
Cases (n) | Controls (s) | Expression change | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Author, year | Gene function | mRNA | EC | EU | EU | NE | Type studied | E | S | Refs. |
Matsuzaki, 2004 | Cell proliferation and apoptosis | PDGFRA | 12 | – | 12 | – | DIE | – | ↑ | ( |
Matsuzaki, 2006 | 12 | – | 12 | – | OE | – | ↑ | ( |
||
Matsuzaki, 2004 | PKCβ1 | 12 | – | 12 | – | DIE | – | ↑ | ( |
|
Matsuzaki, 2006 | 12 | – | 12 | – | OE | – | ↑ | ( |
||
Matsuzaki, 2004 | JAK1 | 12 | – | 12 | – | DIE | – | ↑ | ( |
|
Matsuzaki, 2006 | 12 | – | 12 | – | OE | – | ↑ | ( |
||
Matsuzaki, 2004 | Sprouty2 | 12 | – | 12 | – | DIE | – | ↑ | ( |
|
Matsuzaki, 2006 | 12 | – | 12 | – | DIE | – | ↑ | ( |
||
Matsuzaki, 2004 | MKK7 | 12 | – | 12 | – | DIE | – | ↑ | ( |
|
Gaeje, 2007 | WNT7A | 20 | – | 8 | – | NR | ↑ | ↑ | ( |
|
Matsuzaki, 2005 | MSP | 26 | – | 23 | – | DIE | ↑ | – | ( |
|
Matsuzaki, 2005 | 14 | – | 26 | – | DIE | ↑ | – | ( |
||
Matsuzaki, 2005 | RON | 12 | – | – | 12 | DIE | ↑ |
– | ( |
|
Matsuzaki, 2005 | SOS | 12 | – | – | 12 | DIE | ↑ |
– | ( |
|
Matsuzaki, 2005 | 14-3-3 protein eta | 12 | – | – | 12 | DIE | ↑ |
– | ( |
|
Matsuzaki, 2005 | uPAR | 12 | – | – | 12 | DIE | ↑ |
– | ( |
|
Matsuzaki, 2005 | KSR | 12 | – | – | 12 | DIE | – | ↑ |
( |
|
Matsuzaki, 2005 | PI3K p85 regulatory | 12 | – | – | 12 | DIE | – | ↑ |
( |
|
subunit α | ||||||||||
Matsuzaki, 2004 | Pain pathway | TrKB | 12 | – | 12 | – | DIE | ↑ | – | ( |
Matsuzaki, 2007 | 12 |
– | – | 12 |
DIE | ↑ | – | ( |
||
Matsuzaki, 2004 | MOR | 12 | – | 12 | – | DIE | – | ↑ | ( |
|
Matsuzaki, 2006 | 12 | – | 12 | – | OE | – | ↑ | ( |
||
Matsuzaki, 2007 | 12 |
– | – | 12 |
DIE | – | ↑ | ( |
||
Matsuzaki, 2004 | 5HTT | 12 | – | 12 | – | DIE | – | ↑ | ( |
|
Matsuzaki, 2006 | Hormones and steroids | Aromatase | 16 | 30 | – | 24 | DIE | ↑ | – | ( |
Matsuzaki, 2006 | 17β-HSD2 | – | 30 | – | 24 | DIE | ↑ | – | ( |
|
Matsuzaki, 2006 | 12 | – | 12 | – | OE | ↑ | – | ( |
||
Matsuzaki, 2004 | HSP90A | 12 | – | 12 | – | DIE | ↑ | – | ( |
|
Matsuzaki, 2004 | COUP-TF2 | 12 | – | 12 | – | DIE | ↑ | – | ( |
|
Matsuzaki, 2006 | 12 | – | 12 | – | OE | ↑ | – | ( |
||
Matsuzaki, 2004 | PGE2EP3 | 12 | – | 12 | – | DIE | ↑ | – | ( |
|
Matsuzaki, 2006 | 12 | – | 12 | – | OE | ↑ | – | ( |
||
Matsuzaki, 2009 | Adhesion and implantation | HOXA10 | – | 62 | – | 20 | OE+DIE+PE | – | ↑ |
( |
Matsuzaki, 2010 | E-cadherin | – | 151 | – | 51 | NR | ↑ |
– | ( |
|
Matsuzaki, 2010 | β-catenin | – | 151 | – | 51 | NR | ↑ |
– | ( |
|
Matsuzaki, 2010 | MMP-7 | 137 | – | 50 | DIE | ↑ | – | ( |
||
Wu, 2006 | DNA methylation | DNMT1 | 13 | 10 | 8 | OE+DIE+PE | ↑ | – | ( |
|
Wu, 2006 | DNMT3A | 13 | 10 | 8 | OE+DIE+PE | ↑ | – | ( |
||
Wu, 2006 | DNMT3B | 13 | 10 | 8 | OE+DIE+PE | ↑ | – | ( |
In the late secretory phase;
from GnRH agonist– and progestin-treated patients;
from EC in untreated patients;
in infertile patients. EC, ectopic endometrium; EU, eutopic endometrium; NE, normal endometrium; E, epithelial glandular cell; S, stromal cell; DIE, deep infiltrating endometriosis; OE, ovarian endometriosis; PE, peritoneal endometriosis; NR, not recorded; PDGFRA, platelet-derived growth factor receptor alpha; PKC, protein kinase C; JAK, Janus kinase; MKK7, mitogen-activated protein kinase kinase 7; MSP, major sperm protein; RON, macrophage-stimulating protein receptor; SOS, Son of sevenless; Upar, Urokinase receptor; KSR, kinase suppressor of RAS; PI3K, phosphoinositide-3 kinase; TrKb, tropomyosin receptor kinase B; MOR, middle operon regulator; 5HTT, serotonin transporter gene; 17β-HSD2, 17β-hydroxysteroid dehydrogenase type 2; HSP, heat shock protein; COUP-TF2, chicken ovalbumin upstream promoter transcription factor 2.