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Erschienen in: Reproductive Biology and Endocrinology 1/2018

Open Access 01.12.2018 | Research

Altered miRNA profile in testis of post-cryptorchidopexy patients with non-obstructive azoospermia

verfasst von: Dongdong Tang, Zhenyu Huang, Xiaojin He, Huan Wu, Dangwei Peng, Li Zhang, Xiansheng Zhang

Erschienen in: Reproductive Biology and Endocrinology | Ausgabe 1/2018

Abstract

Background

Cryptorchidism is one of the most common causes of non-obstructive azoospermia (NOA) leading to male infertility. Despite various medical approaches been utilised, many patients still suffer from infertility. MicroRNAs (miRNAs) play vital roles in the progress of spermatogenesis; however, little is known about the miRNA expression profile in the testes. Therefore, the miRNA profile was assessed in the testis of post-cryptorchidopexy patients.

Methods

Three post-cryptorchidopexy testicular tissue samples from patients aged 23, 26 and 28 years old and three testis tissues from patients with obstructive azoospermia (controls) aged 24, 25 and 36 years old were used in this study. Next-generation sequencing (NGS) was used to perform the miRNA expression profiling. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays were subsequently used to confirm the results of several randomly-selected and annotated miRNAs.

Results

A series of miRNAs were found to be altered between post-cryptorchidopexy testicular tissues and control tissues, including 297 downregulated and 152 upregulated miRNAs. In the subsequent qRT-PCR assays, the expression levels of most of the selected miRNAs (9/12, P < 0.05) were consistent with the results of NGS technology. Furthermore, signal transduction, adaptive immune response and biological regulation were associated with the putative target genes of the differentially-expressed miRNAs via GO analysis. In addition, oxidative phosphorylation, Parkinson’s disease and ribosomal pathways were shown to be enriched using KEGG pathway analysis of the differentially-expressed genes.

Conclusions

This study provides a global view of the miRNAs involved in post-cryptorchidopexy testicular tissues as well as the altered expression of miRNAs compared to control tissues, thus confirming the vital role of miRNAs in cryptorchidism.
Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12958-018-0393-3) contains supplementary material, which is available to authorized users.
Dongdong Tang and Zhenyu Huang contributed equally to this work.
Abkürzungen
HE
Hematoxylin-eosin
miRNAs
MicroRNAs
NGS
Next-generation sequencing
NOA
Non-obstructive azoospermia
OA
Obstructive azoospermia
qRT-PCR
Quantitative real-time reverse transcription-polymerase chain reaction

Background

Male factors account for approximately 50% of infertility cases, which affect 10–15% of couples around the world [1]. Although most cases of male infertility are idiopathic with no known etiological factor, some causes (i.e. varicocele, sexual dysfunction etc.) are known [2]. Among these causes, cryptorchidism is a relatively common anomaly in the male genitalia that affects approximately 2–4% of male infants. Despite various medical approaches (i.e. surgical operations and hormone administration) being applied for years, many patients still suffer from infertility [3, 4], and little is known about the clear mechanism of spermatogenesis arrest in these patients.
Spermatogenesis is a complex process consisted of three phases including mitotic, meiotic and haploid processes [5]. These cellular events require highly regulated spatiotemporal expression of specific protein-coding genes, especailly at the post-transcriptional levels [6]. MicroRNAs (miRNAs) are a series of small noncoding RNAs that negatively regulate gene expression after transcription [7]. Research has shown that miRNAs play crucial roles in spermatogenesis [5, 6, 813]; for example, Lian et al. identified a series of altered miRNAs in patients with non-obstructive azoospermia (NOA) using microarray technology. These identified 154 significantly downregulated and 19 upregulated miRNAs indicated the important role of miRNAs in spermatogenesis [10]. It was reported that during mouse testicular development, up-regulation of miR-449 coincided with initiation of meiotic, and miR-449 was predominantly expressed in spermatocytes and spermatids during adult spermatogenesis. Furthermore, Cdc20b/miR-449 cluster activity was documented to be cooperatively mediated by CREMT and SOX5 during postnatal testes development [5]. Later on, Comazzetto et al. have identified the miR-34 family consisted of miR-34b/c and miR-449a/b/c as upregulated from late meiosis to sperm stage. miR-34b/c and miR-449 deletion led to sterility due to abnormal spermatozoa production with reduced motility [11]. With regards to the effects of miRNAs in cryptorchidism, Duan et al. found that miR-210, a significantly upregulated miRNA in patients with NOA, was also highly expressed in patients with cryptorchidism [12]. In addition, Moritoki et al. demonstrated that miR-135a was downregulated in unilateral undescended testes in a rat model of cryptorchidism [13].
Although some miRNAs were shown to be involved in the regulation of spermatogenesis in patients with cryptorchidism, no studies have yet investigated miRNA expression in the testis of post-cryptorchidopexy patients with NOA. Therefore this study investigated the miRNA profile in the testis of post-cryptorchidopexy patients and aimed to provide a platform to expound the mechanism of spermatogenesis arrest in post-cryptorchidopexy patients with NOA.

Methods

Ethics statement

Three patients (23, 26 and 28 years old) who underwent cryptorchidopexy but were still experiencing NOA, as well as three patients (24, 25 and 36 years old) suffering from obstructive azoospermia (OA) signed informed consent and approved the use of their tissues for research purposes. The local medical ethics committee approved this study.

Clinical specimen collection

Testes tissues were collected by testicular biopsy from all six subjects between July 2017 and January 2018 at the Reproductive Medicine Center, First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China). For post-cryptorchidopexy patients, all cases were bilateral. Case one was 23 years old and underwent the operation 1 year ago, case two was 26 years old and underwent the operation 18 years ago and case three was 28 years old and underwent the operation 12 years ago. Testes samples were frozen at − 80 °C in RNAlater (Ambion, USA) immediately after surgery. Haematoxylin and eosin (HE) staining and the Johnson score system were used to assess testicular spermatogenic function.

Construction of a smRNA library and next-generation sequencing (NGS)

Total RNA was extracted from the six samples using TRIzol (Life Technologies, USA) and was used to construct miRNA libraries using the NEBNext® Multiplex Small RNA Library Prep Set (Illumina®) according to the manufacturer’s instructions. Sequencing was performed on a Hiseq X (Illumina) using the HiSeq X Reagent Kit v2.

Data analyses and novel miRNA exploration

Data were analysed according to previously-reported methods. Known miRNAs were identified by mapping reads to miRBase (version 21.0) in Homo sapiens, whilst nonmatched reads were subsequently aligned against other noncoding RNAs within the Ensembl database [14]. The remaining nonannotated sequences were selected for alignment with the integrated human transcriptome to explore novel miRNAs. All hairpin-like structures containing unclassified smRNA reads (no less than 45 reads) were predicted using miRDeep2 [15] following the criteria described previously [16].

Bioinformatic analyses for miRNAs with differential expression patterns

The target genes of the differentially-expressed miRNAs in the two groups were predicted using TargetScan [17] and miRanda [18]. Enriched GO terms and KEGG pathway analysis was subsequently applied to predict the target genes of miRNAs with differential expression patterns in the two groups of specimens.

QRT-PCR verification for altered miRNA expression

cDNA synthesis was performed using a PrimeScript RT reagent kit following the manufacturer’s instructions (Takara, Japan). The abundance of individual miRNAs was subsequently assessed via an Applied Biosystems 7500 PCR System (Applied Biosystems) using SYBR Premix Ex Taq II (Tli RNaseH Plus, Takara) under optimised reaction conditions. The specific reverse transcription and qPCR primers for all miRNAs are listed in Additional file 1. The processes were performed in accordance with the protocols supplied by the manufacturers. Briefly, for qPCR, triplicate reactions were performed at 95 °C for 10 min, and the subsequent 40 amplification cycles were conducted at 95 °C for 15 s and 60 °C for 60 s. Meanwhile, 18S rRNA was used as an internal normalised control. Relative miRNA abundances were calculated using 2−△△Ct (threshold cycle) formula, where △Ct = CtmiRNA − Ct18S rRNA and △△Ct = (△Ctpost-cryptorchidopexy − △Ctobstructive azoospermia). The miRNA concentration differences between post-cryptorchidopexy and control tissues were analysed using unpaired t-tests. P < 0.05 indicated a statistically significant difference.

Results

Histopathological characteristics of post-cryptorchidopexy testicular tissue and control tissue

To clarify the histopathological characteristics of the post-cryptorchidopexy testicular tissue (hereafter referred to as ‘cryptorchid tissue’) and control tissue (hereafter referred to as ‘normal tissue’), HE staining and the Johnson scoring system were used to assess the function of spermatogenesis (Fig. 1). The Johnson scores were 3, 3 and 3 in cryptorchid tissues, which indicated maturation arrest, and 9, 9 and 10 in normal control tissues, which indicated normal spermatogenesis.

Comprehensive overview of whole genome smRNAs in cryptorchid and normal tissues

All smRNAs [18–32 nucleotides (nt)] acquired from cryptorchid and normal tissues were deep sequenced by NGS. A total of 19,931,698 (out of 21,212,215) and 20,243,124 (out of 21,524,351) sequence reads that aligned to the human genome sequence dataset were obtained in the cryptorchid and normal tissues, respectively. MiRNAs accounted for 85.5% and 71.19% in cryptorchid and normal tissues, respectively (Fig. 2).
The most abundant of these smRNAs in cryptorchid tissue were 21 nt in length, and these smRNAs were more abundant than the 22-nt and 23-nt RNAs which were in second and third place, respectively. However, the most abundant smRNAs in normal tissue were 22 nt in length, and these were more abundant than the 21-nt and 23-nt RNAs which were in second and third place, respectively (see Additional file 2).
Understanding the distribution pattern of miRNA genes may help to elucidate their roles, therefore the chromosomal locations of miRNA genes were evaluated. In cryptorchid tissue, most miRNA genes were located on chromosomes X, 9, 3 and 21. Similarly, in normal tissues, most miRNA genes were located on chromosome X, 15, 9 and 5 (see Additional file 3).

Features of the most abundant miRNAs in cryptorchid and normal tissues

The NGS results were used to compile a list of the 20 most abundant and known miRNAs in cryptorchid tissue and the 10 most abundant and novel miRNAs in normal tissue. In cryptorchid tissue, miR-514a-3p, miR-143-3p, miR-26a-5p, miR-99a-5p, miR-202-5p, miR-509-3-5p, miR-10b-5p, miR-508-3p, let-7 g-5p and let-7f-5p were the most abundant known miRNAs. In normal tissue, miR-514a-3p, miR-143-3p, miR-26a-5p, miR-509-3-5p, miR-99a-5p, miR-202-5p, miR-10b-5p, let-7f-5p, miR-508-3p and let-7 g-5p were the most abundant known miRNAs (Table 1). Detailed information is shown in Table 1. Of the 10 most abundant novel miRNAs, only one was different between cryptorchid and normal tissues. Detailed information is shown in Table 2.
Table 1
The top 20 most abundant known miRNAs expressed in cryptorchid and normal tissues
miRNA name
Cryptorchid
miRNA name
Control
Reads count
Normalized reads count
Reads count
Normalized reads count
hsa-miR-514a-3p
2,313,282
109,499
hsa-miR-514a-3p
1,008,914
98,435
hsa-miR-143-3p
864,140
45,306
hsa-miR-143-3p
677,433
65,245
hsa-miR-26a-5p
829,953
46,035
hsa-miR-26a-5p
407,763
39,613
hsa-miR-99a-5p
705,575
38,041
hsa-miR-509-3-5p
392,074
37,711
hsa-miR-202-5p
616,580
30,770
hsa-miR-99a-5p
346,310
33,871
hsa-miR-509-3-5p
593,363
28,054
hsa-miR-202-5p
258,855
25,790
hsa-miR-10b-5p
428,806
24,984
hsa-miR-10b-5p
248,352
24,218
hsa-miR-508-3p
303,445
14,625
hsa-let-7f-5p
154,806
15,116
hsa-let-7 g-5p
296,741
15,472
hsa-miR-508-3p
153,631
15,059
hsa-let-7f-5p
266,118
14,707
hsa-let-7 g-5p
151,745
14,851
hsa-let-7a-5p
265,013
14,644
hsa-let-7a-5p
137,886
13,412
hsa-miR-21-5p
248,959
12,378
hsa-miR-21-5p
132,197
12,931
hsa-miR-509-5p
194,212
9293
hsa-miR-148a-3p
119,576
11,671
hsa-miR-148a-3p
188,828
10,645
hsa-miR-100-5p
103,191
10,071
hsa-miR-125b-5p
172,432
9013
hsa-miR-125b-5p
93,627
9134
hsa-miR-100-5p
169,375
9160
hsa-miR-27b-3p
92,637
8927
hsa-miR-199a-3p
154,971
8592
hsa-miR-509-5p
81,898
8124
hsa-miR-27b-3p
144,132
7610
hsa-miR-126-3p
79,980
7687
hsa-let-7i-5p
140,689
7772
hsa-miR-125a-5p
72,130
7013
hsa-let-7b-5p
112,327
6040
hsa-miR-34c-5p
69,568
6885
hsa-miR-125a-5p
107,449
5594
hsa-let-7i-5p
66,915
6560
Table 2
The list of top 10 most abundant novel miRNAs expressed in cryptorchid and normal tissues
Cryptorchid tissues
miRNA ID
Mature Sequence
Reads count
Location of novel miRNA precusor
chrX_47246
AUUGACACUUCUGUGAGUAGA
2,280,438
chrX:146366172..146366230:-
chr12_27425
UUCAAGUAAUCCAGGAUAGGCU
826,714
chr12:58218403..58218462:-
chr3_5958
UUCAAGUAAUCCAGGAUAGGCU
826,558
chr3:38010903..38010964:+
chr21_44054
AACCCGUAGAUCCGAUCUUGU
693,017
chr21:17911420..17911480:+
chrX_47235
UACUGCAGACGUGGCAAUCAUG
592,879
chrX:146341178..146341235:-
chr10_23103
UUCCUAUGCAUAUACUUCUUU
586,995
chr10:135061041..135061097:-
chr5_9937
UGAGAUGAAGCACUGUAGCUC
534,462
chr5:148808506..148808561:+
chr2_3766
UACCCUGUAGAACCGAAUUUGU
428,617
chr2:177015056..177015117:+
chrX_47228
UGAUUGUAGCCUUUUGGAGUAGA
298,225
chrX:146318462..146318520:-
chr3_7283
UGAGGUAGUAGUUUGUACAGUU
295,643
chr3:52302295..52302373:-
Normal tissues
miRNA ID
Mature Sequence
Reads count
Location of novel miRNA precusor
chrX_47246
AUUGACACUUCUGUGAGUAGA
996,346
chrX:146366172..146366230:-
chr5_9937
UGAGAUGAAGCACUGUAGCUC
419,103
chr5:148808506..148808561:+
chr12_27425
UUCAAGUAAUCCAGGAUAGGCU
405,817
chr12:58218403..58218462:-
chr3_5958
UUCAAGUAAUCCAGGAUAGGCU
405,621
chr3:38010903..38010964:+
chrX_47235
UACUGCAGACGUGGCAAUCAUG
391,755
chrX:146341178..146341235:-
chr21_44054
AACCCGUAGAUCCGAUCUUGU
340,009
chr21:17911420..17911480:+
chr2_3766
UACCCUGUAGAACCGAAUUUGU
248,236
chr2:177015056..177015117:+
chr10_23103
UUCCUAUGCAUAUACUUCUUU
246,558
chr10:135061041..135061097:-
chr9_18744
UGAGGUAGUAGAUUGUAUAGUU
154,925
chr9:96938634..96938712:+
chr3_7283
UGAGGUAGUAGUUUGUACAGUU
151,154
chr3:52302295..52302373:-

Differential expression of miRNAs between cryptorchid and normal tissues

As described previously by Zhang et al. [16], miRNAs were considered to be significantly differentially expressed between cryptorchid and normal tissues if they were altered by at least two-fold with P < 0.05 on the t-test. The results showed that 449 miRNAs were significantly differentially expressed in cryptorchid tissue (Fig. 3). Of these, 297 were downregulated and 152 were upregulated compared to normal tissue. The 30 most downregulated and upregulated known miRNAs are listed in Tables 3 and 4, respectively.
Table 3
A collection of the top 30 most downregulated known miRNAs detected by deep sequencing in cryptorchid tissues
MiRNA name
baseMean
log2FoldChange
lfcSE
stat
p
Adjust p
hsa-miR-3663-5p
41.936
−4.426
0.624
−7.089
1.35E-12
2.39E-10
hsa-miR-1233-3p
25.216
−4.227
0.679
−6.225
4.79E-10
1.84E-08
hsa-miR-552-5p
66.556
−4.055
0.563
−7.195
6.24E-13
1.21E-10
hsa-miR-449b-5p
392.523
−3.972
0.496
−8.001
1.23E-15
5.26E-13
hsa-miR-7153-5p
108.897
− 3.812
0.634
−6.010
1.84E-09
5.18E-08
hsa-miR-122-5p
525.785
−3.790
0.562
−6.741
1.57E-11
1.60E-09
hsa-miR-552-3p
65.189
−3.760
0.562
−6.680
2.38E-11
2.31E-09
hsa-miR-449a
5575.001
−3.740
0.511
−7.317
2.52E-13
5.97E-11
hsa-miR-122-3p
4.738
−3.722
1.011
−3.679
0.00023
0.0016
hsa-miR-34b-5p
123.524
−3.688
0.558
−6.610
3.84E-11
3.56E-09
hsa-miR-449c-5p
2234.173
−3.637
0.465
−7.816
5.42E-15
1.93E-12
hsa-miR-34c-5p
39,328.272
−3.553
0.440
−8.060
7.58E-16
5.26E-13
hsa-miR-449c-3p
7.961
−3.441
0.902
−3.812
0.00014
0.0011
hsa-miR-375
491.449
−3.408
0.362
−9.416
4.68E-21
9.99E-18
hsa-miR-3663-3p
37.612
−3.385
0.676
−5.001
5.68E-07
9.63E-06
hsa-miR-7159-5p
20.897
−3.259
0.705
−4.618
3.87E-06
5.29E-05
hsa-miR-449b-3p
142.460
−3.212
0.610
−5.262
1.42E-07
2.75E-06
hsa-miR-4700-5p
4.985
−3.208
0.951
−3.370
0.00075
0.0043
hsa-miR-522-3p
121.036
−3.153
0.465
−6.768
1.30E-11
1.46E-09
hsa-miR-1273a
38.566
−3.118
0.508
−6.135
8.47E-10
2.44E-08
hsa-miR-1295a
11.735
−3.075
0.760
−4.041
5.31E-05
0.0005
hsa-miR-34b-3p
1137.731
−2.970
0.516
−5.753
8.72E-09
2.16E-07
hsa-miR-1283
139.436
−2.798
0.488
−5.731
9.95E-09
2.41E-07
hsa-miR-3150b-3p
3.547
−2.768
0.991
−2.791
0.0052
0.020
hsa-miR-4423-3p
16.582
−2.702
0.755
−3.578
0.00035
0.0023
hsa-miR-6507-5p
7.696
−2.698
0.811
−3.325
0.00088
0.0049
hsa-miR-7154-5p
406.827
−2.646
0.981
−2.697
0.0070
0.025
hsa-miR-517c-3p
95.074
−2.639
0.386
−6.832
8.37E-12
9.92E-10
hsa-miR-3925-3p
10.324
−2.613
0.735
−3.553
0.00038
0.0025
hsa-miR-515-5p
84.007
−2.600
0.379
−6.856
7.04E-12
8.84E-10
Table 4
A collection of the top 30 most upregulated known miRNAs detected by deep sequencing in cryptorchid tissues
MiRNA name
baseMean
log2FoldChange
lfcSE
stat
p
Adjust p
hsa-miR-7151-3p
6.026
2.634
0.892
2.953
0.0031
0.014
hsa-miR-376a-2-5p
10.918
2.202
0.724
3.042
0.0023
0.011
hsa-miR-1224-5p
17.708
2.193
0.615
3.565
0.00036
0.0024
hsa-miR-1299
187.854
1.958
0.426
4.600
4.22E-06
5.73E-05
hsa-miR-142-5p
697.547
1.898
0.583
3.255
0.0011
0.0060
hsa-miR-543
1281.559
1.869
0.450
4.152
3.29E-05
0.00036
hsa-miR-487a-3p
80.564
1.865
0.591
3.155
0.0016
0.0079
hsa-miR-584-3p
19.666
1.829
0.562
3.254
0.0011
0.0060
hsa-miR-665
18.416
1.798
0.710
2.534
0.011
0.036
hsa-miR-134-3p
29.541
1.778
0.598
2.975
0.0029
0.013
hsa-miR-369-3p
500.851
1.692
0.432
3.916
8.99E-05
0.00082
hsa-miR-377-3p
96.245
1.665
0.551
3.023
0.0025
0.011
hsa-miR-33a-5p
28.103
1.664
0.550
3.025
0.0025
0.011
hsa-miR-376a-3p
112.0733
1.602
0.436
3.704
0.00021
0.0015
hsa-miR-758-3p
520.1303
1.589
0.439
3.620
0.00029
0.0020
hsa-miR-654-3p
4175.568
1.587
0.388
4.095
4.22E-05
0.00044
hsa-miR-134-5p
2747.859
1.558
0.424
3.675
0.00024
0.0017
hsa-miR-889-3p
740.3619
1.552
0.468
3.312
0.00093
0.0052
hsa-miR-127-3p
40,871.646
1.548
0.392
3.955
7.65E-05
0.00071
hsa-miR-1185-1-3p
161.457
1.539
0.506
3.039
0.0024
0.011
hsa-miR-1185-2-3p
38.541
1.534
0.587
2.614
0.0089
0.030
hsa-miR-154-5p
267.267
1.516
0.346
4.385
1.16E-05
0.00014
hsa-miR-381-3p
7512.422
1.511
0.382
3.957
7.57E-05
0.00070
hsa-miR-127-5p
768.176
1.511
0.401
3.765
0.00017
0.0013
hsa-miR-337-5p
44.570
1.510
0.439
3.437
0.00059
0.0036
hsa-miR-379-3p
262.022
1.508
0.401
3.756
0.00017
0.0013
hsa-miR-136-3p
937.135
1.506
0.389
3.868
0.00011
0.00096
hsa-miR-376c-3p
327.216
1.492
0.402
3.713
0.00020
0.0015
hsa-miR-495-3p
884.797
1.443
0.390
3.696
0.00022
0.0016
hsa-miR-376b-5p
24.828
1.442
0.590
2.445
0.014
0.045

Validating the altered expression level of miRNAs by qRT-PCR

QRT-PCR was performed to validate the altered miRNA expression. Among these deregulated miRNAs, we firstly selected two well-established spermatogenesis-associated miRNAs, miR-449a and miR-34c-5p [5, 11]. Additionally, to better proving the accuracy of NGS, the other validated miRNAs were picked from the non-top 30 most deregulated known miRNAs (see Additional file 4 and Additional file 5), so that the relatively small fold changes could be validated. According to the previous studies, ten miRNAs were picked for qRT-PCR validation randomly [16, 19]. Eventually, a total of 12 differentially-expressed miRNAs (seven upregulated and five downregulated) were selected for qRT-PCR analysis. The results showed that the expression levels of most miRNAs (9/12; P < 0.05) were consistent with the results of NGS technology. Detailed information is shown in Fig. 4.

GO enrichment analysis of differentially-expressed genes in cryptorchid and normal tissues

After predicting the target genes of differentially-expressed miRNAs in cryptorchid and normal tissues, GO enrichment analysis was conducted. The 10 most enriched GO terms, including signal transduction and adaptive immune response, are shown in Table 5.
Table 5
Top 30 most enriched GO terms for predicted targets of differentially expressed miRNAs between cryptorchid and normal tissues
GO number
Term*
GO process
Ratio in study
Ratio in pop
p
GO:0007165
BP
signal transduction
19.68%
23.63%
1.04E-05
GO:0002250
BP
adaptive immune response
0.70%
1.83%
1.56E-05
GO:0050789
BP
regulation of biological process
47.95%
52.34%
4.14E-05
GO:0050794
BP
regulation of cellular process
45.10%
49.34%
7.66E-05
GO:0008150
BP
biological_process
78.42%
81.73%
8.25E-05
GO:0065007
BP
biological regulation
51.05%
55.25%
8.51E-05
GO:0006956
BP
complement activation
0.25%
0.98%
0.000114
GO:0006958
BP
complement activation, classical pathway
0.20%
0.88%
0.000134
GO:0048518
BP
positive regulation of biological process
21.53%
25.03%
0.00014
GO:0050776
BP
regulation of immune response
3.95%
5.72%
0.000229
GO:0044425
CC
membrane part
28.37%
34.86%
1.03E-10
GO:0005886
CC
plasma membrane
17.68%
23.38%
1.11E-10
GO:0031224
CC
intrinsic component of membrane
23.23%
29.30%
2.06E-10
GO:0016021
CC
integral component of membrane
22.68%
28.69%
2.24E-10
GO:0005575
CC
cellular_component
84.22%
88.01%
1.38E-07
GO:0005794
CC
Golgi apparatus
3.35%
5.84%
1.42E-07
GO:0005840
CC
ribosome
2.20%
1.09%
7.38E-06
GO:0000139
CC
Golgi membrane
1.85%
3.41%
1.92E-05
GO:0044459
CC
plasma membrane part
9.84%
12.81%
1.93E-05
GO:0004872
MF
receptor activity
5.39%
8.48%
5.53E-08
GO:0060089
MF
molecular transducer activity
5.39%
8.48%
5.53E-08
GO:0005179
MF
hormone activity
1.55%
0.54%
6.49E-08
GO:0004871
MF
signal transducer activity
5.79%
8.78%
2.25E-07
GO:0038023
MF
signaling receptor activity
4.45%
7.13%
3.21E-07
GO:0099600
MF
transmembrane receptor activity
4.25%
6.85%
4.09E-07
GO:0003823
MF
antigen binding
0.50%
1.75%
4.18E-07
GO:0004888
MF
transmembrane signaling receptor activity
4.15%
6.63%
9.33E-07
GO:0032553
MF
ribonucleotide binding
6.34%
8.94%
1.10E-05
GO:0003674
MF
molecular_function
77.82%
81.17%
7.79E-05
*BP Biological process; CC Cellular component; MF Molecular function

KEGG pathway analysis of differentially-expressed genes in cryptorchid and normal tissues

After GO analysis, KEGG pathway enrichment analysis was performed. A total of five KEGG pathways were enriched, including oxidative phosphorylation, Parkinson’s disease, Ribosomal pathways, Huntington’s disease and Alzheimer’s disease. The results are presented in Table 6.
Table 6
KEGG pathway analysis for predicted target genes of differentially expressed miRNAs between cryptorchid and normal tissues
Pathway ID
Description
GeneRatio
BgRatio
p
Adjust p
GeneName
hsa00190
Oxidative phosphorylation
28/664
133/7297
1.80E-05
0.0053
ATP5G2;COX6C;SDHD;COX7A2L;
COX8C;ATP6V1D
hsa05012
Parkinson’s disease
28/664
142/7297
6.29E-05
0.0093
ATP5G2;COX6C;SDHD;UBB;UBE2L6;COX7A2L;GNAL;COX8C
hsa03010
Ribosome
29/664
154/7297
0.0001112
0.0110
MRPL16;RPL38;RPS4X;MRPL35;
RPS6;MRPS18C;RPL26;RPS27L
hsa05016
Huntington’s disease
33/664
193/7297
0.0002633
0.0187
ATP5G2;COX6C;UCP1;SDHD;
POLR2J3;COX7A2L;COX8C;POLR2K
hsa05010
Alzheimer’s disease
30/664
171/7297
0.0003147
0.0187
ATP5G2;COX6C;CASP12;SDHD;
PPP3CC;COX7A2L;LPL;COX8C

Discussion

As one of the most common congenital defects in newborn boys, cryptorchidism influences male fertility and increases the risk of testicular cancer. Reductions in seminiferous tubules and germ cells are common histological changes in cryptorchid testis [20]. Despite surgery being recommended for many patients with cryptorchidism, the success of orchidopexy depends on the timing of the procedure and the position of the testis: some may not benefit from cryptorchidopexy [21, 22]. Although research has identified some biological processes involved in spermatogenic arrest in cryptorchid testis (i.e. significant apoptotic changes in germ cells), the causative roles of genes in spermatogenic arrest or apoptosis remain unclear [2326]. This is the first study to investigate the possible mechanisms of spermatogenic arrest in cryptorchid testes by assessing the miRNA profiles in post-cryptorchidopexy testes.
Many rodent and primate models were developed to identify altered miRNAs in cryptorchid testis. For example, Duan et al. established a mouse model of cryptorchidism and showed that miR-210 was highly expressed in cryptorchid testes compared with control testes. Moreover, they showed that this miRNA regulated spermatogenesis by inhibiting the expression of NR1D2 [12]. Moritoki et al. compared the miRNA expression profiles of unilateral undescended testes with contralateral descended testes in a rat model of cryptorchidism using microarray analysis. These authors found that only miR-135a expression was lower in unilateral undescended testes and that its target, FoxO1, played essential roles in stem cell maintenance [13]. Furthermore, Duan et al., also found that miR-210 was upregulated in human cryptorchidism, thus suggesting a vital role for miRNAs in humans [12]. In this study, 297 downregulated and 152 upregulated miRNAs were identified in post-cryptorchidopexy testicular tissue compared with normal testis tissue. However, miR-210 was not significantly altered, which may be due to the different types of human cryptorchid tissue. For example, Duan et al. used cryptorchid testis tissue obtained during the cryptorchidopexy, whilst this study used post-cryptorchidopexy testicular tissue. Some miRNA expression levels may change after the operation.
Despite the insights gained into cryptorchidism over the years, the mechanism of spermatogenesis arrest in patients with this disease remains largely elusive. Germ cell apoptosis is commonly seen at the histological level in cryptorchid testes. Yin et al. revealed that cryptorchidism induced germ cell apoptosis in an experimental mouse model via p53-dependent and p53-independent pathways [23]. Liu et al. also found that the Hsf1/Phlda1 pathway participated in primary spermatocyte apoptosis in surgery-induced cryptorchid testes of rats [27]. The expression of many apoptosis-related miRNAs was also shown to be altered in post-cryptorchidopexy testicular tissues. It was reported that miR-299-5p could modulate apoptosis through autophagy in neurons and ameliorate the cognitive capacity of APPswe/PS1dE9 mice [28]. In addition, miR-299-5p was significantly upregulated in post-cryptorchidopexy testicular tissue. Similar results were also found for miR-217, miR-206 etc. Li et al. also found that miR-217 could regulate apoptosis by targeting TNFSF11 in human podocyte cells [29]. This study also identified a significant downregulation of miR-217 in post-cryptorchidopexy testicular tissue. Similarly, miR-206 was significantly upregulated in post-cryptorchidopexy testicular tissue and was shown to promoted cell apoptosis in Legg–Calvé–Perthes disease [30].

Conclusions

In summary, miRNA expression in post-cryptorchidopexy testicular tissue was profiled using NGS and compared with that of OA men with normal spermatogenesis. Several signalling pathways that are likely to be involved in spermatogenesis arrest in these patients were addressed. The results provide an important platform for future investigations into the roles of miRNAs in the progression of cryptorchidism as well as therapeutic targets to help these patients recover fertility. However, the comprehensive modulating behaviours of genes remain unclear, therefore determining the target genes and regulatory networks of these differentially-expressed miRNAs is essential in future investigations.

Acknowledgements

We thank all subjects who provided the tissues for this study.

Funding

This study was funded by the the National Natural Science Foundation of China (No. 81370749).

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Written informed consent was obtained from all patients and this study was approved by the Ethics Review Board of The First Affiliated Hospital of Anhui Medical University.
Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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Literatur
1.
Zurück zum Zitat Matzuk MM, Lamb DJ. Genetic dissection of mammalian fertility pathways. Nat Cell Biol. 2002;4(Suppl):s41–9.PubMed Matzuk MM, Lamb DJ. Genetic dissection of mammalian fertility pathways. Nat Cell Biol. 2002;4(Suppl):s41–9.PubMed
2.
Zurück zum Zitat Practice Committee of the American Society for Reproductive Medicine. Diagnostic evaluation of the infertile male: a committee opinion. Fertil Steril. 2015;103:e18–25. Practice Committee of the American Society for Reproductive Medicine. Diagnostic evaluation of the infertile male: a committee opinion. Fertil Steril. 2015;103:e18–25.
3.
Zurück zum Zitat Kolon TF, Herndon CD, Baker LA, Baskin LS, Baxter CG, Cheng EY, et al. Evaluation and treatment of cryptorchidism: AUA guideline. J Urol. 2014;192:337–45.CrossRefPubMed Kolon TF, Herndon CD, Baker LA, Baskin LS, Baxter CG, Cheng EY, et al. Evaluation and treatment of cryptorchidism: AUA guideline. J Urol. 2014;192:337–45.CrossRefPubMed
4.
Zurück zum Zitat Barthold JS, Gonzalez R. The epidemiology of congenital cryptorchidism, testicular ascent and orchiopexy. J Urol. 2003;170:2396–401.CrossRefPubMed Barthold JS, Gonzalez R. The epidemiology of congenital cryptorchidism, testicular ascent and orchiopexy. J Urol. 2003;170:2396–401.CrossRefPubMed
5.
Zurück zum Zitat Bao J, Li D, Wang L, Wu J, Hu Y, Wang Z, et al. MicroRNA-449 and microRNA-34b/c function redundantly in murine testes by targeting E2F transcription factor-retinoblastoma protein (E2F-pRb) pathway. J Biol Chem. 2012;287:21686–98.CrossRefPubMedPubMedCentral Bao J, Li D, Wang L, Wu J, Hu Y, Wang Z, et al. MicroRNA-449 and microRNA-34b/c function redundantly in murine testes by targeting E2F transcription factor-retinoblastoma protein (E2F-pRb) pathway. J Biol Chem. 2012;287:21686–98.CrossRefPubMedPubMedCentral
6.
Zurück zum Zitat Bouhallier F, Allioli N, Lavial F, Chalmel F, Perrard MH, Durand P, et al. Role of miR-34c microRNA in the late steps of spermatogenesis. RNA. 2010;16:720–31.CrossRefPubMedPubMedCentral Bouhallier F, Allioli N, Lavial F, Chalmel F, Perrard MH, Durand P, et al. Role of miR-34c microRNA in the late steps of spermatogenesis. RNA. 2010;16:720–31.CrossRefPubMedPubMedCentral
7.
Zurück zum Zitat Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–97.CrossRefPubMed Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–97.CrossRefPubMed
8.
Zurück zum Zitat Tang D, Huang Y, Liu W, Zhang X. Up-regulation of microRNA-210 is associated with spermatogenesis by targeting IGF2 in male infertility. Med Sci Monit. 2016;22:2905–10.CrossRefPubMedPubMedCentral Tang D, Huang Y, Liu W, Zhang X. Up-regulation of microRNA-210 is associated with spermatogenesis by targeting IGF2 in male infertility. Med Sci Monit. 2016;22:2905–10.CrossRefPubMedPubMedCentral
9.
10.
Zurück zum Zitat Lian J, Zhang X, Tian H, Liang N, Wang Y, Liang C, et al. Altered microRNA expression in patients with non-obstructive azoospermia. Reprod Biol Endocrinol. 2009;7:13.CrossRefPubMedPubMedCentral Lian J, Zhang X, Tian H, Liang N, Wang Y, Liang C, et al. Altered microRNA expression in patients with non-obstructive azoospermia. Reprod Biol Endocrinol. 2009;7:13.CrossRefPubMedPubMedCentral
11.
Zurück zum Zitat Comazzetto S, Di Giacomo M, Rasmussen KD, Much C, Azzi C, Perlas E, et al. Oligoasthenoteratozoospermia and infertility in mice deficient for miR-34b/c and miR-449 loci. PLoS Genet. 2014;10:e1004597.CrossRefPubMedPubMedCentral Comazzetto S, Di Giacomo M, Rasmussen KD, Much C, Azzi C, Perlas E, et al. Oligoasthenoteratozoospermia and infertility in mice deficient for miR-34b/c and miR-449 loci. PLoS Genet. 2014;10:e1004597.CrossRefPubMedPubMedCentral
13.
Zurück zum Zitat Moritoki Y, Hayashi Y, Mizuno K, Kamisawa H, Nishio H, Kurokawa S, et al. Expression profiling of microRNA in cryptorchid testes: miR-135a contributes to the maintenance of spermatogonial stem cells by regulating FoxO1. J Urol. 2014;191:1174–80.CrossRefPubMed Moritoki Y, Hayashi Y, Mizuno K, Kamisawa H, Nishio H, Kurokawa S, et al. Expression profiling of microRNA in cryptorchid testes: miR-135a contributes to the maintenance of spermatogonial stem cells by regulating FoxO1. J Urol. 2014;191:1174–80.CrossRefPubMed
16.
Zurück zum Zitat Zhang L, Wei P, Shen X, Zhang Y, Xu B, Zhou J, et al. MicroRNA expression profile in penile Cancer revealed by next-generation small RNA sequencing. PLoS One. 2015;10:e0131336.CrossRefPubMedPubMedCentral Zhang L, Wei P, Shen X, Zhang Y, Xu B, Zhou J, et al. MicroRNA expression profile in penile Cancer revealed by next-generation small RNA sequencing. PLoS One. 2015;10:e0131336.CrossRefPubMedPubMedCentral
18.
Zurück zum Zitat John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. Human MicroRNA targets. PLoS Biol. 2004;2:e363. John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. Human MicroRNA targets. PLoS Biol. 2004;2:e363.
19.
Zurück zum Zitat Zhou Y, Wang X, Zhang Y, Zhao T, Shan Z, Teng W. Circulating microrna profile as a potential predictive biomarker for early diagnosis of spontaneous abortion in patients with subclinical hypothyroidism. Front Endocrinol (Lausanne). 2018;9:128.CrossRef Zhou Y, Wang X, Zhang Y, Zhao T, Shan Z, Teng W. Circulating microrna profile as a potential predictive biomarker for early diagnosis of spontaneous abortion in patients with subclinical hypothyroidism. Front Endocrinol (Lausanne). 2018;9:128.CrossRef
20.
Zurück zum Zitat Agoulnik AI, Huang Z, Ferguson L. Spermatogenesis in cryptorchidism. Methods Mol Biol. 2012;825:127–47.CrossRefPubMed Agoulnik AI, Huang Z, Ferguson L. Spermatogenesis in cryptorchidism. Methods Mol Biol. 2012;825:127–47.CrossRefPubMed
21.
Zurück zum Zitat Taran I, Elder JS. Results of orchiopexy for the undescended testis. World J Urol. 2006;24:231–9.CrossRefPubMed Taran I, Elder JS. Results of orchiopexy for the undescended testis. World J Urol. 2006;24:231–9.CrossRefPubMed
22.
Zurück zum Zitat Hadziselimovic F, Thommen L, Girard J, Herzog B. The significance of postnatal gonadotropin surge for testicular development in normal and cryptorchid testes. J Urol. 1986;136:274–6.CrossRefPubMed Hadziselimovic F, Thommen L, Girard J, Herzog B. The significance of postnatal gonadotropin surge for testicular development in normal and cryptorchid testes. J Urol. 1986;136:274–6.CrossRefPubMed
23.
Zurück zum Zitat Yin Y, DeWolf WC, Morgentaler A. Experimental cryptorchidism induces testicular germ cell apoptosis by p53-dependent and -independent pathways in mice. Biol Reprod. 1998;58:492–6.CrossRefPubMed Yin Y, DeWolf WC, Morgentaler A. Experimental cryptorchidism induces testicular germ cell apoptosis by p53-dependent and -independent pathways in mice. Biol Reprod. 1998;58:492–6.CrossRefPubMed
24.
Zurück zum Zitat Yin Y, Stahl BC, DeWolf WC, Morgentaler A. P53 and Fas are sequential mechanisms of testicular germ cell apoptosis. J Androl. 2002;23:64–70.CrossRefPubMed Yin Y, Stahl BC, DeWolf WC, Morgentaler A. P53 and Fas are sequential mechanisms of testicular germ cell apoptosis. J Androl. 2002;23:64–70.CrossRefPubMed
25.
Zurück zum Zitat Mu X, Liu Y, Collins LL, Kim E, Chang C. The p53/retinoblastoma-mediated repression of testicular orphan receptor-2 in the rhesus monkey with cryptorchidism. J Biol Chem. 2000;275:23877–83.CrossRefPubMed Mu X, Liu Y, Collins LL, Kim E, Chang C. The p53/retinoblastoma-mediated repression of testicular orphan receptor-2 in the rhesus monkey with cryptorchidism. J Biol Chem. 2000;275:23877–83.CrossRefPubMed
26.
Zurück zum Zitat Li W, Bao W, Ma J, Liu X, Xu R, Wang RA, et al. Metastasis tumor antigen 1 is involved in the resistance to heat stress-induced testicular apoptosis. FEBS Lett. 2008;582:869–73.CrossRefPubMed Li W, Bao W, Ma J, Liu X, Xu R, Wang RA, et al. Metastasis tumor antigen 1 is involved in the resistance to heat stress-induced testicular apoptosis. FEBS Lett. 2008;582:869–73.CrossRefPubMed
27.
Zurück zum Zitat Liu F, Xu ZL, Qian XJ, Qiu WY, Huang H. Expression of Hsf1, Hsf2, and Phlda1 in cells undergoing cryptorchid-induced apoptosis in rat testes. Mol Reprod Dev. 2011;78:283–91.CrossRefPubMed Liu F, Xu ZL, Qian XJ, Qiu WY, Huang H. Expression of Hsf1, Hsf2, and Phlda1 in cells undergoing cryptorchid-induced apoptosis in rat testes. Mol Reprod Dev. 2011;78:283–91.CrossRefPubMed
28.
Zurück zum Zitat Zhang Y, Liu C, Wang J, Li Q, Ping H, Gao S, et al. MiR-299-5p regulates apoptosis through autophagy in neurons and ameliorates cognitive capacity in APPswe/PS1dE9 mice. Sci Rep. 2016;6:24566.CrossRefPubMedPubMedCentral Zhang Y, Liu C, Wang J, Li Q, Ping H, Gao S, et al. MiR-299-5p regulates apoptosis through autophagy in neurons and ameliorates cognitive capacity in APPswe/PS1dE9 mice. Sci Rep. 2016;6:24566.CrossRefPubMedPubMedCentral
29.
Zurück zum Zitat Li J, Liu B, Xue H, Zhou QQ, Peng L. miR-217 is a useful diagnostic biomarker and regulates human podocyte cells apoptosis via targeting tnfsf11 in membranous nephropathy. Biomed Res Int. 2017;2017:2168767.PubMedPubMedCentral Li J, Liu B, Xue H, Zhou QQ, Peng L. miR-217 is a useful diagnostic biomarker and regulates human podocyte cells apoptosis via targeting tnfsf11 in membranous nephropathy. Biomed Res Int. 2017;2017:2168767.PubMedPubMedCentral
30.
Zurück zum Zitat Luo J, Han J, Li Y, Liu Y. Downregulated SOX9 mediated by miR-206 promoted cell apoptosis in Legg-calve-Perthes disease. Oncol Lett. 2018;15:1319–24.PubMed Luo J, Han J, Li Y, Liu Y. Downregulated SOX9 mediated by miR-206 promoted cell apoptosis in Legg-calve-Perthes disease. Oncol Lett. 2018;15:1319–24.PubMed
Metadaten
Titel
Altered miRNA profile in testis of post-cryptorchidopexy patients with non-obstructive azoospermia
verfasst von
Dongdong Tang
Zhenyu Huang
Xiaojin He
Huan Wu
Dangwei Peng
Li Zhang
Xiansheng Zhang
Publikationsdatum
01.12.2018
Verlag
BioMed Central
Erschienen in
Reproductive Biology and Endocrinology / Ausgabe 1/2018
Elektronische ISSN: 1477-7827
DOI
https://doi.org/10.1186/s12958-018-0393-3

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