Aims We previously found colonic crypts with asymmetric fission bordering regenerating ulcers in ulcerative colitis (UC). The present objective was to assess the frequency of asymmetric crypt-fission in colectomy specimens from patients with long-lasting UC.
Methods H&E-stained sections from seven colectomies from patients with UC without dysplasia or carcinoma were investigated. Symmetric fission was characterised by branched colon crypts showing ≥2 identical crypts, whereas asymmetric fission exhibited branched colon crypt portraying ≥2 dissimilar crypts, differing in diameter, length and/or shape.
Results The number of crypts in fission in the 89 sections was 3586; of those, 2930 (81.7%) were asymmetric and the remaining 656 (18.3%), symmetric. Out of 927 vertically-cut crypts (in well-oriented sections), 912 (98.4%) were asymmetric, and the remaining 14 (1.6%), symmetric, and out 2660, cross-cut (transected) crypts in fission, 2018 (75.9%) were asymmetric and the remaining 642 (24.1%), symmetric.
Conclusion Crypt fission is rarely found in the normal colon in adults. Symmetric crypt fission found in UC is possibly triggered by a compensatory homeostatic mechanism of crypt production in mucosal areas replaced by chronic inflammation. But asymmetric crypt fission is a pathological aberration that affects crypts in patients with a particular predisposition to develop mucosal dysplasia. It is suggested that this previously unattended histological parameter be included in the pathological descriptions of colectomy specimens from patients with UC.
- inflammatory bowel diseases
Data availability statement
All data relevant to the study are included in the article.
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The normal mucosa of the colon in humans and rodents is composed of an assemblage of mucosal invaginations called glands or crypts that are closely aligned as parallel ‘test tubes’ vertical to the surface epithelium and to the muscularis mucosae.1–3 The production of a new crypt is caused by symmetric bifurcation or fission.4 Pari passu with postnatal growth in infant rats, crypts replicate by symmetric fission, beginning at their base and progress upwards until two identical individual crypts are finally formed.4 In the normal colon mucosa of adults, however, crypts in fission are rarely found.1–3 In 1972, Morson5 found crypt budding and branching in rectal biopsies from patients with ulcerative colitis (UC) in remission. In 1984, we analysed rectal biopsies from 61 patients with UC in remission.6 Transversely cut crypts displayed crypt dichotomy (crypt fission); it was referred to as the ‘back-to-back sign’. In addition, crypts with lateral buddings were found.5 More recently, Tan et al 7 found that about 90% of the dissected single colon crypts from C57BL/6 mice replicated ‘buds’ growing near the bottom, a phenomenon called asymmetric bud fission. On the other hand, Bomman and Fields8 claimed that budding and bifurcations only develop in the dysplastic crypts of colorectal adenomas.
In a previous work, we quantified colonic crypts with non-dysplastic epithelium although with corrupted shapes (CCS) in three colectomy specimens from patients with UC.9 Remarkably, 43% of the 70 CCS crypts surrounding mucosal ulcerations displayed asymmetric fission.9
The aim of the present communication was to investigate in more detail the frequency of asymmetric and symmetric crypt fission found in a cohort of colectomy specimens from patients with UC.
Materials and methods
Seven colectomy specimens from patients clinically diagnosed as extensive UC without low-grade and high-grade dysplasia, adenoma (focal dysplastic neoplasia) or carcinoma were investigated. Samples corresponding to the left and transverse colon displayed moderate-to-severe degrees of inflammation.
Tissue samples were obtained from 11 different segments. Sections were stained with H&E. A review of the sections was carried out on a standard Nikon light microscope (Surbiton, UK), carrying ×10 ocular lenses and a ×10 Plan Apo objective (aperture 0.45). At that magnification, the entire field of vision was 2 mm in diameter.
In this study, normal innominate grooves (characterised by a single central crypt lumen into which several horizontal or oblique subsidiary crypts in pairs opened) were disregarded.
Symmetric crypt fission
Asymmetric crypt fission
In well-oriented crypts
Asymmetric crypt-fission disclosing ≥2 crypts of different diameters, lengths and/or irregular shapes (figures 2 and 3).
Innominate grooves: In the normal mucosa, these grooves are described in well-oriented crypts as multiple crypts in a ‘mirror image’ opening into a common lumen, generally found within the superficial one-third of the mucosa.2 Hence, normal innominate grooves display a single central crypt lumen into which several sideways, horizontal or oblique subsidiary crypts open. It should be pointed out, however, that the two or more sideways crypts in the grooves might not always be identical in shape and length, particularly in UC. Therefore, it is not always easy to distinguish innominate grooves from asymmetric crypts in well-oriented sections in UC. Examples of this limitation are shown in figure 2B and E.
It should be stressed that unquestionable innominate grooves were disregarded in the present work.
In transected (transversely cross-cut) crypts: Two or more back-to-back asymmetric colon crypt-fissions separated by a thin epithelial rim.
Parameters registered in individual sections
The length of individual sections (in mm).
The number of vertical crypts with symmetric fission.
The number of vertical crypts with asymmetric fission, including epithelial buds.
The number of transected (cross-cut) crypts with symmetric fission.
The number of transected (cross-cut) crypts in asymmetric fission, including epithelial buds.
The non-parametric Kruskal-Wallis test was applied to compare the difference between groups. Statistical significance was defined as p<0.05.
Since the study is retrospective and the specimens were coded and studied anonymised, The Regional Ethical Review Board in Stockholm approved that informed consent from the patients was not needed.
Number and length of tissue sections
Table 1 shows that 89 sections were reviewed (mean 12.7 sections/colectomy, range 11 to 18 sections/colectomy). The total length of the sections investigated in the seven colectomy specimens was 3991 mm (mean 570.1 mm, range 235 to 1007 mm).
The number of crypts with asymmetrical and symmetrical fission
Out of the total number of 3586 crypts in fission recorded in the seven colectomy specimens in table 1, 2930 (81.7%) were asymmetrical and the remaining 656 (18.3%) symmetrical (p<0.05).
The number of vertical crypts and transected crypts in fission
The number of vertically cut crypts exhibiting asymmetrical and symmetrical fission
Table 2 also shows that out of a total number of 927 vertically cut crypts recorded in sections from the seven colectomies, 912 (98.4%) were asymmetrical and the remaining 14 (1.6%) were symmetrical (p<0.05).
The number of cross-cut crypts disclosing asymmetrical and symmetrical fission
Out of a total number of 2660 cross-cut (transected) crypts in fission recorded in the seven colectomy specimens in table 2, 2018 (75.9%) were asymmetrical and the remaining 642 (24.1%) were symmetrical (p<0.05).
In this communication, we report a high frequency of crypts with symmetric and asymmetric fission in colectomy specimens from patients with UC without dysplasia or carcinoma. This contrasts with the normal colon mucosa of adults where crypts in fission are rarely found.1 2 . In this regard, we previously studied the histological sections from 22 normal colonic segments, proximal or distal, from surgically removed colonic adenocarcinoma.10 Using a 10× ocular and a 10× objective (aperture 0.45), the number of crypts present in 15 continuous fields of view, corresponding to 30 mm of colonic mucosa, was registered for each of the control segments. A total of 8580 crypts present in the 15 continuous fields of view/segment (or 30 mm/segment) were registered in the 22 controls (mean=390 crypts/segment, range=382 to 408). Crypts with normal shapes lined with normal epithelium were found in 19 colonic segments, and in the remaining three segments, occasional crypts with irregular architecture (mean=3.7, range=2 to 5) were recorded. No crypts in symmetric or asymmetric fission were found.10 Humphries and Wright11 found only occasional crypts in symmetric fission in the normal adult colon; crypts in asymmetric fission were not reported.
The increased frequency of crypts in symmetric fission in UC might have been triggered by a compensatory homeostatic mechanism of crypt production in areas having colonic crypts destroyed by the inflammatory process. Asymmetric crypt fission was detected in vertical crypts in well-oriented sections and in transected crypts in transversely cross-cut sections. Notably, 82% of the crypts in fission were asymmetrical, with some displaying two, three or more irregular crypt fission. The term multiasymmetric fission is proposed for the latter option.
It should be mentioned that the high number of crypts in symmetric and asymmetric fission in Case 4 (table 1) was found in the colectomy with the highest number of sections, and consequently, with the longest length (mm) examined.
In 1998, Wasan et al microdissected individual crypts from patients with familial fission transversely (FAP).4 Crypts with small epithelial bud formations were called abnormally-dividing and regarded as asymmetric buds. Epithelial buds were found in the upper lateral portion of the crypts in 80% and at the base of the crypts in 20%. Crypts fission with asymmetric buds were more frequently seen in FAP patients than in controls.4 Years later, Tan et al dissected and isolated crypts from the colon of C57BL/6 mice.7 Using quantitative 3D immunofluorescence of colon crypts and confocal image analysis technique, the authors postulated that colon crypts can be generated by asymmetrical epithelial buds emerging near the bottom of the crypts (called Stage 1).7 Colonic crypt budding rates decreased with the increased age of the animals. Tan et al 7 found heterogeneity in the subcellular distribution of E-cadherin and β-catenin in asymmetrical buds. In contrast to Wasan et al, 4 Tan et al 7 found no symmetrical epithelial buddings in the middle or in the upper part of the isolated crypts. More recently, Bomman and Fields8 claimed that colonic crypts develop histological abnormalities only after becoming dysplastic, that is, during the formation of adenomas. Buddings and bifurcations in dysplastic crypts started not at the crypt bottom but somewhere along the crypt axis.8 More recently, Bruens et al 12 claimed that colonic crypt fission can be re-assembled by crypt fusion in mice. This phenomenon was recently confirmed in humans by Baker.13 Observation by Bruens and Baker underpin the multifaceted mechanism of colon crypt replication.
According to Tan et al, 7 bud formation in isolated colon crypts suggested that the constraint forces from the surrounding lamina propria prevented crypt bud formation in histological sections from the normal mucosa. If this is the case, then the presence of crypt buds of asymmetric crypt fission in colectomies from UC patients would imply that the constraint forces in the microenvironment of the lamina propria in histological sections might be partly or totally abrogated by the ongoing chronic inflammation.
The repopulation of the crypts is synchronised by the stem cells.7 8 11 13–16 The stem cell niche, located at the crypt base, maintains cell number homeostasis in the colonic crypts. Wnt signalling controls cell proliferation, differentiation and apoptosis along the crypts; Wnt concentration is high at the bottom of the crypt (where stem cells reside) and low at the top. In contrast, APC concentration is low at the crypt bottom and high at the top (where differentiated cells reside).4 8 14 Wnt signalling has been linked to crypt fission in infant rats and is required for the production of colon crypts in mice in vitro.14 In addition, Jagan et al 17 demonstrated that the formation of colorectal crypts is regulated by phosphatase and tensin homolog deleted on chromosome 10, a protein encoded by the PTEN gene. Moreover, Georgescu et al 18 found that NHERF1 protein, a Na+/H+exchanger regulatory factor, controls human colonic gland morphogenesis.
Against that knowledge, the pertinent questions are:
Why asymmetric crypt fission develops in the colon mucosa of patient with UC? In this context, it should be pointed out that crypts with normal epithelium but with architectural distortions accruing below the dysplastic compartment of sporadic conventional colon adenomas thrive with relocated proliferating cell (PC) domains and p53 upregulated cell.19 Rationally, the rearrangement of PC domains in the crypts with normal epithelium but with architectural distortions might be the result of the relocation of the cells that fuel the production of PC, namely the stem cells.13 15 Since the relocation of PC domains in the crypts with architectural distortions is not an accidental event, the following relevant questions are: Why do the PC domains have to be relocated? Are the crypts with architectural distortions running their own cell replication agenda? What molecular signals have upset the natural symmetric PC homeostasis in those crypts? Is this because the PC relocation in crypts with architectural distortions is crucial for the escalation of cell proliferation in the neoplastic epithelium on top? Ongoing studies of cell proliferation in crypts with architectural distortions in UC might disclose whether crypts in asymmetric fission also thrive with relocated PC domains.
Could not the yet known mutated molecular signals generated from the severely inflamed lamina propria microenvironment trigger the development of asymmetric crypt fission?
What is the role played by intracryptal neutrophil abscesses in the development of asymmetric crypt fission (figure 4)? Does the intracryptal destruction of the mucosa by the intracryptal abscesses trigger crypt fission asymmetry? If that is the case, then crypts with asymmetric fission without abscesses might be the reflection of crypts with a past history of intracryptal abscesses.
Are the putative relocated stem cells in crypts with asymmetric fission more susceptible to oncogenic mutations?
What role is played by the mutagenic bacteria found in UC20–22 in the development of asymmetric crypts fission?
Are crypts with asymmetric fission triggered by UC medication before colectomy?
From the above, it is evident that the present survey raises many questions but provides but few answers. Notwithstanding, asymmetric crypt fission is a pathological aberration that affects crypts in patients with a particular predisposition to develop mucosal dysplasia. One possibility is, that should not be disregarded, mutations of the normal Wnt signalling16 of NHERF118 and PTEN signalling17 might be operating in the colonic crypts of patients with UC, resulting in the development of asymmetrical crypt fission.
It should be pointed out that crypts in asymmetric fission can also be found in:
The non-dysplastic compartment of polypoid and non-polypoid conventional colon adenomas.19
signalling serrated lesions.23
In this regard, out of 47 endoscopic biopsies in patients with Crohn’s colitis, 43 had crypts in asymmetric fission. The total number of crypts in asymmetric fission found in the 43 cases was 201 (mean 4.7 asymmetric crypts per biopsy, range 1 to 15) (Rubio CA, Schmidt PT, unpublished).
Further research is necessary to ascertain whether the putative mutated asymmetric crypt fission has any bearing on the predisposition of some patients to develop epithelial dysplasia, such as those with UC.24 It is suggested that this previously unattended histological parameter be included in the pathological descriptions of colectomy specimens from patients with UC.
Take home messages
Crypts in fission are rarely found in the normal colon mucosa of adults.
Crypts with symmetric fission and asymmetric fission often occur in colectomy specimens from patient with ulcerative colitis (UC).
The high frequency of symmetric crypt fission in UC might be the result of a compensatory homeostatic mechanism of crypt production in areas having numerous crypts destroyed by the inflammatory process.
Asymmetric crypt fission, a pathological aberration that affects crypts in patients with a particular predisposition to develop mucosal dysplasia, was frequently found in this material.
It is suggested that this previously unattended histological parameter be included in the pathological descriptions of colectomy specimens from patients with UC.
Data availability statement
All data relevant to the study are included in the article.
This study was approved by The Regional Ethical Review Board in Stockholm (no. 2011/252-31/2 and 2018/980-32).
Handling editor Dhirendra Govender.
Contributors CAR collected the initial data, collected and organised the data on the pathological findings, and wrote the original draft. PTS obtained the permission of The Regional Ethical Review Board in Stockholm, revised the original draft and introduced valuable suggestions.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer-reviewed.
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