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Molecular profiling of follicular fluid microRNAs in young women affected by Hodgkin lymphoma

Open AccessPublished:August 15, 2021DOI:https://doi.org/10.1016/j.rbmo.2021.08.007

      Abstract

      Research question

      Treatments for Hodgkin lymphoma have improved but one of their common effects is gonadal toxicity, which contributes to fertility damage of patients and induces temporary or irreversible loss of fertility. Could micro-RNA (miRNA) expression profiles in follicular fluid be influenced by Hodgkin lymphoma? Could their alteration affect molecular pathways involved in follicle growth and oocyte maturation?

      Design

      miRNA expression profile was investigated in follicular fluid samples from young women affected by Hodgkin lymphoma compared with healthy controls by NanoString technology. Bioinformatic analysis was used to verify miRNA involvement in follicle development and miRNA deregulation with Hodgkin lymphoma in a larger cohort of follicular fluid samples was confirmed by real-time quantitative polymerase chain reaction.

      Results

      Thirteen miRNAs are deregulated in Hodgkin lymphoma samples compared with controls and are involved in molecular pathways related to cancer, gametogenesis and embryogenesis. Among them, let-7b-5p, miR-423-5p, miR-503-5p, miR-574-5p and miR-1303 are implicated in biological processes related to follicle development and oocyte maturation. Let-7b-5p holds the central position in the regulatory network of miRNA–mRNA interactions, has the highest number of mRNA target genes shared with the other differentially expressed miRNAs and is significantly downregulated in Hodgkin lymphoma follicular fluid samples.

      Conclusions

      These data led us to question the potential influence of miRNA deregulation on oocyte quality. Further studies are needed to verify the reproductive potential of young patients with Hodgkin lymphoma before starting chemotherapy protocols and an adequate protocol of fertility preservation needs to be guaranteed.

      KEYWORDS

      Introduction

      Hodgkin lymphoma is one of the most frequent cancers among young adults in developed countries. Advanced early diagnosis and treatments have lowered its mortality rate, resulting in a high cure rate and a long survival time for patients affected by this malignancy (
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      ).
      Short cycles of chemotherapy combined with radiotherapy and combination chemotherapy are generally used to treat patients with early and advanced stage Hodgkin lymphoma, respectively (
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      Should Response-Adapted Therapy Now Be the Standard of Care for Advanced Hodgkin's Lymphoma?.
      ). These treatments could affect the quality of life of survivor patients. One of their most common effects is gonadal toxicity. Although treatments for Hodgkin lymphoma are effective, they may contribute to fertility damage of patients, inducing temporary or irreversible loss of fertility (
      • Melo A.S.
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      ).
      Chemotherapy and radiotherapy expose women affected by Hodgkin lymphoma to an increased risk of early menopause and ovarian follicle depletion (
      • Morgan S.
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      • Wallace W.H.
      • Spears N.
      How do chemotherapeutic agents damage the ovary?.
      ). Although the diagnostic and therapeutic advances in oncology have improved the quality of life of young cancer patients, treatments may affect the ovarian reserve, causing a major risk of ovarian failure and infertility (
      • Spears N.
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      ). The extension of the damage to the ovary depends on several factors, such as type and dose of chemotherapy, radiotherapy dose, fractionation scheme, irradiation field and the ovarian reserve before treatment (
      • Wallace W.H.
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      ). Anti-Müllerian hormone (AMH) and antral follicle count (AFC) are considered helpful predictors of ovarian damage after cancer treatment (
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      ).
      To avoid complications in future fertility, selected treatment strategies are required. Young women especially should be informed about the possibility of fertility preservation before starting cancer treatments (
      • De Vos M.
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      • Woodruff T.K.
      Fertility preservation in women with cancer.
      ). Currently, fertility preservation techniques for woman are oocyte cryopreservation, IVF for embryo cryopreservation and cryopreservation of ovarian tissue (
      • Melo A.S.
      • Paula C.T.V.
      • Rufato M.A.F.
      • Rufato M.
      • Rodrigues J.K.
      • Ferriani R.A.
      • Barreto J.
      Fertility optimization in women with cancer: from preservation to contraception.
      ). In young women affected by haematological cancers, oocyte cryopreservation seems to be the best option as most of them have not yet planned whether and when to create a family, and the preservation of ovarian tissue may reintroduce residual malignant cells (
      • De Vos M.
      • Smitz J.
      • Woodruff T.K.
      Fertility preservation in women with cancer.
      ). It seems clear that many factors should be considered when approaching future reproduction.
      Some studies have reported full-term pregnancies after the thawing of vitrified oocytes; however, results are still insufficient (
      • Kato K.
      • Ochi M.
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      • Asada Y.
      • Ohishi H.
      • Osada H
      • Yoshida H.
      A multi-centre, retrospective case series of oocyte cryopreservation in unmarried women diagnosed with haematological malignancies.
      ). Information on the number of women who use their frozen oocytes, pregnancy and live birth rates should be collected to ensure the usefulness of fertility preservation techniques (https://www.eshre.eu/en).
      Haematological malignancies, including Hodgkin lymphoma, are diffuse tumours and could affect a woman's reproductive potential resulting in reduced ovarian reserve, lower oocyte quality, or both, even before starting chemotherapy (
      • Fabbri R.
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      • Magnani V.
      • Arpinati M.
      • Battaglia C.
      • Paradisi R.
      • Venturoli S.
      Follicle features in adolescent and young adult women with Hodgkin's disease prior to chemotherapy: a preliminary report.
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      • Fehm T.
      • von Wolff M.
      • Soekler M.
      • Huebner S.
      • Henes J.
      • Henes M.
      Centers of Ferti, P. N.
      Reduced pretreatment ovarian reserve in premenopausal female patients with Hodgkin lymphoma or non-Hodgkin-lymphoma–evaluation by using antimullerian hormone and retrieved oocytes.
      ;
      • Lekovich J.
      • Lobel A.L.S.
      • Stewart J.D.
      • Pereira N.
      • Kligman I.
      • Rosenwaks Z.
      Female patients with lymphoma demonstrate diminished ovarian reserve even before initiation of chemotherapy when compared with healthy controls and patients with other malignancies.
      ;
      • Paradisi R.
      • Vicenti R.
      • Macciocca M.
      • Seracchioli R.
      • Rossi S.
      • Fabbri R.
      High cytokine expression and reduced ovarian reserve in patients with Hodgkin lymphoma or non-Hodgkin lymphoma.
      ;
      • Ozdemir Z.N.
      • Bozdag S.C.
      Hematological Malignancies and Fertility.
      ). To the best of our knowledge, however, no research has been conducted on follicular microenvironment and oocyte quality in young women with Hodgkin lymphoma.
      Cell-free microRNAs (miRNAs) have been extensively studied in Hodgkin lymphoma, and different studies have reported significant expression differences between cancer and unaffected patients (
      • Jones K.
      • Nourse J.P.
      • Keane C.
      • Bhatnagar A.
      • Gandhi M.K.
      Plasma microRNA are disease response biomarkers in classical Hodgkin lymphoma.
      ;
      • van Eijndhoven M.A.
      • Zijlstra J.M.
      • Groenewegen N.J.
      • Drees E.E.
      • van Niele S.
      • Baglio S.R.
      • Koppers-Lalic D.
      • van der Voorn H.
      • Libregts S.F.
      • Wauben M.H.
      • de Menezes R.X.
      • van Weering J.R.
      • Nieuwland R.
      • Visser L.
      • van den Berg A.
      • de Jong D.
      • Pegtel D.M.
      Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients.
      ;
      • Cordeiro A.
      • Monzo M
      • Navarro A.
      Non-Coding RNAs in Hodgkin Lymphoma.
      ;
      • Khare D.
      • Goldschmidt N.
      • Bardugo A.
      • Gur-Wahnon D.
      • Ben-Dov I.Z.
      • Avni B.
      Plasma microRNA profiling: Exploring better biomarkers for lymphoma surveillance.
      ). In the same way, follicular fluid miRNA profiles have been associated with the outcome of IVF cycles and with reproductive disorders affecting oocyte quality in human and animal models (
      • Scalici E.
      • Traver S.
      • Mullet T.
      • Molinari N.
      • Ferrieres A.
      • Brunet C.
      • Belloc S.
      • Hamamah S.
      Circulating microRNAs in follicular fluid, powerful tools to explore in vitro fertilization process.
      ;
      • Machtinger R.
      • Rodosthenous R.S.
      • Adir M.
      • Mansour A.
      • Racowsky C.
      • Baccarelli A.A.
      • Hauser R.
      Extracellular microRNAs in follicular fluid and their potential association with oocyte fertilization and embryo quality: an exploratory study.
      ;
      • Martinez R.M.
      • Liang L.
      • Racowsky C.
      • Dioni L.
      • Mansur A.
      • Adir M.
      • Bollati V.
      • Baccarelli A.A.
      • Hauser R.
      • Machtinger R.
      Extracellular microRNAs profile in human follicular fluid and IVF outcomes.
      ). Moreover, alterations of miRNA expression in follicular fluid have also been reported in advanced maternal age (
      • Diez-Fraile A.
      • Lammens T.
      • Tilleman K.
      • Witkowski W.
      • Verhasselt B.
      • De Sutter P.
      • Benoit Y.
      • Espeel M.
      • D'Herde K.
      Age-associated differential microRNA levels in human follicular fluid reveal pathways potentially determining fertility and success of in vitro fertilization.
      ;
      • Battaglia R.
      • Musumeci P.
      • Ragusa M.
      • Barbagallo D.
      • Scalia M.
      • Zimbone M.
      • Lo Faro J.M.
      • Borzi P.
      • Scollo P.
      • Purrello M.
      • Vento E.M.
      • Di Pietro C.
      Ovarian aging increases small extracellular vesicle CD81(+) release in human follicular fluid and influences miRNA profiles.
      ). This evidence supports the hypothesis that follicular fluid miRNAs may represent molecular markers of oocyte quality.
      The aim of the present study was to investigate if follicular fluid miRNA profiles could be influenced by Hodgkin lymphoma and if miRNA expression differences may alter molecular pathways involved in follicular growth and oocyte maturation and, therefore, the quality of the female mature gamete.

      Materials and methods

       Follicular fluid sample collection

      Participants enrolled in this study had been treated with gonadotrophin releasing hormone agonists (triptorelin 0.1 mg/day or buserelin 1 mg/day) until HCG trigger to induce multiple follicular development; ovarian stimulation was then carried out using recombinant FSH and human menopausal gonadotrophin. Stimulation was monitored using serum oestradiol concentrations and ultrasound measurement of follicle number and diameter. Ovulation was induced with 10,000 IU HCG only when follicles had reached a diameter wider than 18 mm and serum oestradiol concentration per follicle reached 150–200 ng/l. Transvaginal ultrasound-guided aspiration of ovarian follicles was carried out 34–36 h after HCG injection. Healthy women whose primary infertility was caused by a male factor were included in the control group; it excluded pathologies that could influence oocyte quality, e.g. endometriosis, polycystic ovaries and ovarian insufficiency. Moreover, heavy smokers and overweight women were excluded from the study. Basic and clinical information for all participants are presented in Table 1.
      Table 1BASIC AND CLINICAL INFORMATION FOR ALL PATIENTS ENROLLED IN THE STUDY
      ParametersWomen with Hodgkin lymphoma (n = 23)Control women (n = 7)P-value (Hodgkin lymphoma versus control)
      Age, years18–2530–34<0.00001
      BMI, kg/m221–2521–250.9
      Recombinant FSH total dose, IU1225–30001625–33250.07
      HMG total dose, IU525–1950450–7500.96
      Follicles with diameters >16 mm11–287–140.02
      Retrieved oocytes15–2811–160.006
      Metaphase II oocytes13–207v120.0007
      Pregnancy
      Pregnancy was defined by both beta-HCG and ultrasounds.
      (%)
      25
      Data are reported as range of values or %. P-values are based on two-sample t-test.
      BMI, body mass index; HMG, human menopausal gonadotrophin.
      a Pregnancy was defined by both beta-HCG and ultrasounds.
      Follicular fluid samples were centrifuged for 20 min at 1000 g at 4°C to remove residual follicular cells and any blood traces; the supernatant was collected and stored at –20°C until its use. Samples with massive blood contamination were excluded from the study and only follicular fluid samples in which nuclear mature oocytes (metaphase II) had been identified were used. The patients signed an informed consent to participate in the research project, which included the use of collected follicular fluid and plasma. The study was exempted from Institutional Review Board approval because patients were included in the IVF programme. Accordingly, no identifiers linked individuals to the samples and no additional treatment or use of personal data were necessary.

       RNA isolation and quantification

      Nanostring protocol requires at least 100 ng of RNA in a final volume of 3 µl. According to this, RNA was isolated separately from two aliquots of 200 µl of the same follicular fluid sample by using the Qiagen miRNeasy Mini Kit (Qiagen, GmbH, Hilden, Germany), according to Qiagen Supplementary Protocol for purification of RNA (including small RNAs) from serum or plasma. In the final step of the procedure, the two aliquots of the same follicular fluid sample were mixed together and precipitated. Specifically, RNA was eluted in 200 µl of RNAse-free water with the addition of 20 µg of UltraPure Glycogen (Thermo Fisher Scientific,Waltham, MA, USA), 0.1 volume of 3 M sodium acetate and 2.5 volumes of ice-cold absolute ethanol and incubated at –80°C overnight. RNA was then centrifuged and washed three times in ice-cold 75% ethanol, resuspended in 7 µl of RNAse-free water and stored at –80°C for further analyses. Total RNA was quantified before and after precipitation by using both a spectrophotometer and the Qubit™ fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Samples showing a 260–280 ratio absorbance of approximately 2.0 were considered.

       High-throughput micro RNA expression analysis

      NanoString nCounter system was used to analyse the expression profile of 800 miRNAs from follicular fluid samples by using the NanoString technology and the nCounter Human v3 miRNA Expression Assay Kits (NanoString Technologies, Seattle, WA, USA), according to the manufacturer's instructions. MiRNA expression profiling was carried out on four follicular fluid samples from young women affected by Hodgkin lymphoma and four young women were used as controls (Table 1).
      Approximately 100 ng of RNA in a final volume of 3 µl was used. Briefly, RNA samples first underwent a miRNA sample preparation step, consisting of annealing of specific tags to target miRNAs, ligation and enzymatic purification allowing the removal of unligated tags. The CodeSet hybridization and the processing of samples were then carried out by using the automated nCounter Prep Station. At this point, samples were purified and immobilized on a sample cartridge for quantification and data collection by using the nCounter Digital Analyzer. Raw data normalization was carried out by using the nSolver 3.0 software and applying the normalization method based on the calculation of geometric mean of the 100 targets with the highest counts, according to the user manual instructions (https://www.nanostring.com/products/analysis-software/nsolver).

       MicroRNA single assay

      Two microlitres of precipitated and concentrated RNA were used for miRNA-specific reverse transcription to obtain miRNA-specific cDNAs, and then analysed by real-time quantitative polymerase chain reaction (RT-qPCR), using TaqMan MicroRNA Assays (Applied Biosystems, Waltham, MA, USA). RT-qPCR reactions were carried out in a final volume of 20 µl, containing 10 µl of TaqMan Universal PCR Master Mix (Applied Biosystems, Waltham, MA, USA), 1 µl of miRNA-specific TaqMan MicroRNA Assay (Applied Biosystems, Waltham, MA, USA), 2 µl of real-time products, and 7 µl of nuclease-free water, by using the 7900HT Fast Real-Time PCR System (Applied Biosystems) as follows: 95°C for 10 min, followed by 40 amplification cycles of 95°C for 15 s and 60°C for 1 min. Validation of miRNA expression was assessed in a cohort of 19 Hodgkin lymphoma samples and three control samples. U6 was used to normalize expression data (
      • Sang Q.
      • Yao Z.
      • Wang H.
      • Feng R.
      • Wang H.
      • Zhao X.
      • Xing Q.
      • Jin L.
      • He L.
      • Wu L.
      • Wang L.
      Identification of microRNAs in human follicular fluid: characterization of microRNAs that govern steroidogenesis in vitro and are associated with polycystic ovary syndrome in vivo.
      ;
      • Feng R.
      • Sang Q.
      • Zhu Y.
      • Fu W.
      • Liu M.
      • Xu Y.
      • Shi H.
      • Xu Y.
      • Qu R.
      • Chai R.
      • Shao R.
      • Jin L.
      • He L.
      • Sun X.
      • Wang L.
      MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro.
      ;
      • Li X.
      • Zhang W.
      • Fu J.
      • Xu Y.
      • Gu R.
      • Qu R.
      • Li L.
      • Sun Y.
      • Sun X.
      MicroRNA-451 is downregulated in the follicular fluid of women with endometriosis and influences mouse and human embryonic potential.
      ).

       MicroRNA function enrichment analyses

      Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene ontology analyses were conducted to deepen the potential biological role of differentially expressed miRNAs. Diana-miRPath v3.0 (http://snf-515788.vm.okeanos.grnet.gr/) was used to select validated mRNA targets retrieved from Tarbase 7.0. The false discovery rate method was applied to select the signalling pathways with a threshold of significance defined by P ≤ 0.05 and a microT threshold of 0.8.
      Differentially expressed miRNA target genes were analysed for gene ontology enrichment by using the Ovarian Kaleidoscope database (http://okdb.appliedbioinfo.net/) to determine the biological process of follicle development and oocyte maturation categories.
      Regulatory network reporting miRNA–mRNA interactions was computed by using the miRNet 2.0 database (https://www.mirnet.ca/miRNet/home.xhtml) and selecting validated target genes from the Tarbase 8.0. After linking the differentially expressed miRNAs with their target mRNAs, they were selected according to follicle development and oocyte maturation biological processes. Target mRNAs interacting with at least two miRNAs were shown in the regulatory network.

       Statistical analysis

      For miRNA profiling analysis, the volcano plot and significance analysis of microarrays (SAM) statistical tests were carried out using MeV (Multi experiment Viewer v4.8.1) software. Differentially expressed miRNAs with statistical significance were screened using the threshold log2 fold change ≥0.5 (log2FC) and P ≤ 0.05 corrected for multiple testing by using Bonferroni method.
      The SAM statistical test was carried out as follows: all statistical tests were computed by applying a two-class unpaired test among log2FC and using a q-value based on 100 permutations; imputation engine: K-nearest neighbours, number of K-nearest neighbours: 10 neighbours. The Benjamini–Hochberg multiple testing correction method for high-throughput analyses using a stringent false discovery rate limit less than 0.05 was applied. Unpaired t-test was applied for differentially expressed miRNA expression validation by using GraphPad Prism 6. Statistical significance was assessed by setting P ≤ 0.05 as cut-off.

      Results

       Basic and clinical information

      The basic and clinical information of women enrolled in the study and the statistical differences between the two groups (women with Hodgkin lymphoma and control women) are presented in Table 1.

       Expression profiling of microRNAs in follicular fluid of women with Hodgkin lymphoma

      High-throughput miRNA expression analysis revealed 27 differentially expressed miRNAs as shown by volcano plot (Figure 1) and 14 differentially expressed miRNAs obtained by SAM analysis. Differentially expressed miRNAs common to both statistical tests were selected for further analyses (Table 2). Seven upregulated miRNAs were selected in Hodgkin lymphoma follicular fluid samples compared with controls (miR-1285-5p, miR-1303, miR-1972, miR-2117, miR-4455, miR-548ah-5p and miR-574-5p) and six downregulated miRNAs in Hodgkin lymphoma follicular fluid samples compared with controls (let-7b-5p, miR-3195, miR-371a-5p, miR-423-5p, miR-4532 and miR-503-5p). Relative expression of differentially expressed miRNAs is shown in Figure 2 (let-7b-5p, P = 0.01; miR-1285-5p, P < 0.001; miR-1303, P = 0.009; miR-1972, P < 0.001; miR-2117, P = 0.002; miR-3195, P = 0.002; miR-371a-5p, P = 0.04; miR-423-5p, P = 0.02; miR-4455, P = 0.005; miR-4532, P = 0.04; miR-503-5p, P = 0.01; miR-548ah-5p, P = 0.01 and miR-574-5p, P = 0.002).
      Figure 1
      Figure 1Volcano plot labelling the most significant microRNAs (miRNAs). Volcano plot showing the differences in fold change (log2FC) of miRNA expression in Hodgkin lymphoma follicular fluid samples compared with controls obtained after data normalization analysis. The x-axis shows differences in log2FC and the y-axis represents the -log10P-value. The horizontal dashed line indicates the threshold for probability of significance (P = 0.05) and the vertical dashed lines set the threshold 0.5 for the difference in fold change of miRNA expression. The green dots represent miRNAs whose expression level is at least 0.5-fold different in Hodgkin lymphoma follicular fluid samples compared with controls, with P < 0.05 corrected for multiple testing by using the Bonferroni method.
      Table 2DIFFERENTIALLY EXPRESSED MICRORNAS IN HODGKIN LYMPHOMA FOLLICULAR FLUID SAMPLES COMPARED WITH CONTROLS
      Diffentially expressed microRNAst-testSAMFold change Hodgkin lymphoma versus controls
      let-7b-5p
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      –3.71
      hmiR-122-5p8.85
      miR-1255b-5p1.97
      miR-1268a–2.14
      miR-1285-5p
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      6.56
      miR-1303
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      3.8
      miR-13231.61
      miR-151a-3p1.91
      miR-1972
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      4.36
      miR-2117
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      2.73
      miR-21-5p–2.13
      miR-22-3p–2.54
      miR-23a-3p–1.55
      miR-3195
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      –2.8
      miR-371a-5p
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      –3.72
      miR-423-5p
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      –3.36
      miR-4455
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      3.66
      miR-450a-5p–2.27
      miR-4532
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      –3.79
      miR-503-5p
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      –4.4
      miR-548ah-5p
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      2.58
      miR-548d-3p2.21
      miR-548d-5p2.71
      miR-548n1.96
      miR-548z+miR-548h-3p2.26
      miR-574-5p
      Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      5.57
      miR-664b-3p2.3
      miR-8901.7
      Differentially expressed miRNAs selected according to volcano plot and significance analysis of microarrays (SAM) statistical tests. The fold change value of each miRNA is reported.
      a Differentially expressed miRNAs common to both statistical tests and chosen for further analyses.
      Figure 2
      Figure 2MiRNA relative expression in follicular fluid samples. Box-and-whisker plots showing the relative expression of the 13 DE miRNAs in Hodgkin lymphoma follicular fluid samples compared with controls. Expression data are represented as log2 normalized counts. Significant P-values corrected for multiple testing by using Benjamini–Hochberg method are indicated (*P < 0.001).

       Functional enrichment analyses

      Differentially expressed miRNA functions were investigated for molecular signalling pathway enrichment and gene ontology classification specific for follicle development and oocyte maturation biological processes.
      Functional enrichment analyses showed that the validated target genes of differentially expressed miRNAs may regulate several signalling pathways involved in fertility and cancer. Most of them are known to be involved in gametogenesis and embryogenesis (
      • Timofeeva A.V.
      • Chagovets V.V.
      • Drapkina Y.S.
      • Makarova N.P.
      • Kalinina E.A.
      • Sukhikh G.T.
      Cell-Free, Embryo-Specific sncRNA as a Molecular Biological Bridge between Patient Fertility and IVF Efficiency.
      ). The most significant signalling pathways were as follows: p53 signalling pathway; pathways in cancer; the TGF-beta signalling pathway; the MAPK signalling pathway; fatty acid metabolism; transcriptional misregulation in cancer; endometrial cancer; the oestrogen signalling pathway; the FoxO signalling pathway; oocyte meiosis; the Hippo signalling pathway; adherens junctions; endocytosis; protein processing in endoplasmic reticulum; cell cycle; and fatty acid biosynthesis (Figure 3).
      Figure 3
      Figure 3Functional enrichment analysis of differentially expressed miRNAs. Functional enrichment analysis of all differentially expressed miRNA target genes using Kyoto Encyclopedia of Genes and Genomes pathway analysis. The x-axis represents the -log10 (P-value).
      Among the 13 differentially expressed miRNAs, let-7b-5p, miR-423-5p, miR-503-5p, miR-574-5p and miR-1303 are known to be involved in oocyte quality, oocyte maturation and ovarian response (see discussion). Among their target genes, 121 out of 5727 were involved in follicle development and oocyte maturation (Table 3).
      Table 3DIFFERENTIALLY EXPRESSED MICRORNA TARGET GENES INVOLVED IN FOLLICLE DEVELOPMENT AND OOCYTE MATURATION
      Gene ontology categoryGene symbol identifiers
      Follicle development (GO_0001541)BIRC2, BRD2, CAMK4, CCND2, CDKN1B, ERMP1, FOSL2, HSPG2, IGF1R, IGF2, IGFBP3, IL1R1, IRS2, ITGA6, JAG1, LAMC1, LRP1, MAP2, NTRK2, OPA1, PAPPA, PDE4D, PTEN, PTPRF, PTX3, SKI, SMAD7, SPHK1, STK4, TAF4, THBS1, VIM
      Oocyte maturation (GO_0001556)ACTB, ACTR3, ADAMTS1, ANLN, AQP3, C3, CALR, CCNT2, CDC25A, CDC25B, CDH2, CDK6, CDKN1A, CENPA, CLTC, CNOT7, CSNK2A1, DCP1A, DICER1, ENO1, ERCC6, EXOC4, EXOSC10, FASTK, FSCN1, GAPDH, GMFB, HDAC2, HSPA8, HUWE1, IMPDH1, ITGB1, ITSN2, JMY, KIF2A, KIF3A, LSM14B, MAD2L1, MAPK1, MAPKAPK2, MCL1, MIS12, MPHOSPH6, MYO10, NOTCH2, PAK3, PBX1, PCNA, PDE3A, PGRMC1, POLR2A, PPP2R1A, PRKAR1A, PRKAR2A, PUM1, PURA, RAP1A, RASA1, RHOB, RICTOR, RIOK1, SALL4, SATB1, SCD, SF1, SLC2A1, SLC4A2, SLC6A9, SLC9A1, SMC1A, SUMO1, TAF9, TGOLN2, TNIK, TOB1, TUBG1, UBR2, UCHL1, VAPB, VPS72, WASF2, WEE1, WNT5A, XBP1, XPO1, YES1, YWHAE, YWHAZ, ZFP36L2
      RNA target genes regulated by the five selected microRNAs and involved in follicle development and oocyte maturation based on gene ontology (GO) computational analysis using the Ovarian Kaleidoscope database.
      Regulatory network analysis revealed that let-7b-5p is the central node of the network having the highest number of target genes (degree 107) that are shared with the other miRNAs in the network (Figure 4, Figure 5).
      Figure 4
      Figure 4Regulatory network of microRNA (miRNA)-target interactions. Regulatory network showing the interaction between let-7b-5p, miR-423-5p, miR-503-5p, miR-574-5p, miR-1303 and their target genes. Black squares represent miRNAs and grey circles represent target genes involved in follicle development and oocyte maturation. Node degree values are reported in the bottom right table of the figure.
      Figure 5
      Figure 5Target genes of differentially expressed miRNAs are involved in follicle development and oocyte maturation. Differentially expressed miRNA target genes involved in follicle development and oocyte maturation are sorted by miRNA-mRNA interaction. Legend shows differentially coloured squares related to each miRNA.

       Let-7b-5p expression in Hodgkin lymphoma follicular fluid samples

      The altered expression of let-7b-5p was confirmed by RT-qPCR in a larger cohort of follicular fluid samples. As reported in Figure 6, let-7b-5p was significantly downregulated in Hodgkin lymphoma follicular fluid samples compared with controls (P = 0.02, unpaired t-test).
      Figure 6
      Figure 6Let-7b-5p is downregulated in Hodgkin lymphoma (HL) follicular fluid samples. Box-and-whisker plots showing the relative expression of let-7b-5p in Hodgkin lymphoma follicular fluid samples compared with controls (CTRL). Expression data are represented as –1*ΔCt values normalized against U6 (P = 0.02).

      Discussion

      Over the years, cancer treatments have become more successful and patient recoveries have increased. It is particularly important in young women to consider the long-term side-effects of cancer treatments that may reduce their quality of life. Infertility represents one of these common effects and, for this reason, different strategies of fertility preservation are now the focus of intense research. In addition, the hope of a future pregnancy after cancer treatment may contribute to a better acceptance of oncological treatment and its adverse effects, and improve patients’ personal experience of cancer (
      • De Vos M.
      • Smitz J.
      • Woodruff T.K.
      Fertility preservation in women with cancer.
      ).
      To date, the current guidelines on female fertility preservation provide clinical recommendations to improve the quality of this practice for many women and young girls (https://www.eshre.eu/en). Suitable stimulation protocols for cancer patients and cryopreservation techniques have only significantly improved in the last few years; for this reason, more data are needed to prove the validity of these options.
      In the present study, a high-throughput miRNA expression analysis was conducted in follicular fluid samples from young women affected by Hodgkin lymphoma. Young women who attended the IVF treatment centre for male infertility problems were used as controls. Significant differences were found between women with Hodgkin lymphoma women and controls in age and retrieved oocytes (Table 1). In fact, the age of women affected by Hodgkin lymphoma is lower than that of controls, although it is known that it is difficult to find women between the ages of 18 and 25 years who have access to an IVF centre without presenting specific problems. In the same way, it is expected that younger women have a better response to the stimulation protocols. In the present study, we are certain that our controls can be defined reproductively as young considering the age and standard parameters shown in Table 1.
      We identified 13 differentially expressed miRNAs in follicular fluid from women with Hodgkin lymphoma compared with controls, indicating that miRNA expression profiles in follicular fluid could be influenced by cancer conditions. In-silico analyses of KEGG pathways and gene ontology analyses revealed that differentially expressed miRNA target genes could regulate molecular signalling pathways involved in cancer, gametogenesis and embryogenesis.
      Among the most significant molecular signalling pathways, PI3K-Akt, MAPK, Hippo and Wnt signalling pathways are known to be involved in protein synthesis, cell survival, migration, invasion, cell cycle progression and cellular proliferation and differentiation (
      • Hers I.
      • Vincent E.E.
      • Tavare J.M.
      Akt signalling in health and disease.
      ;
      • Mo J.S.
      • Park H.W.
      • Guan K.L.
      The Hippo signaling pathway in stem cell biology and cancer.
      ). In particular, the Hippo pathway regulates the balance between differentiation and self-renewal in embryonic stem cells and interacts with other pathways to promote and maintain pluripotency (
      • Varelas X.
      • Sakuma R.
      • Samavarchi-Tehrani P.
      • Peerani R.
      • Rao B.M.
      • Dembowy J.
      • Yaffe M.B.
      • Zandstra P.W.
      • Wrana L.
      • J
      TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal.
      ;
      • Hers I.
      • Vincent E.E.
      • Tavare J.M.
      Akt signalling in health and disease.
      ;
      • Lorthongpanich C.
      • Issaragrisil S.
      Emerging Role of the Hippo Signaling Pathway in Position Sensing and Lineage Specification in Mammalian Preimplantation Embryos.
      ;
      • Ye H.
      • Li X.
      • Zheng T.
      • Hu C.
      • Pan Z.
      • Huang J.
      • Li J.
      • Li W.
      • Zheng Y.
      The Hippo Signaling Pathway Regulates Ovarian Function via the Proliferation of Ovarian Germline Stem Cells.
      ;
      • Kawashima I.
      • Kawamura K.
      Regulation of follicle growth through hormonal factors and mechanical cues mediated by Hippo signaling pathway.
      ;
      • Hsueh A.J.W.
      • Kawamura K.
      Hippo signaling disruption and ovarian follicle activation in infertile patients.
      ).
      The Wnt signalling pathway is involved in cell fate decisions, stem cell maintenance, body-axis determination in vertebrate embryos and gastrulation (
      • Hernandez Gifford J.A.
      The role of WNT signaling in adult ovarian folliculogenesis.
      ;
      • Song J.L.
      • Nigam P.
      • Tektas S.S.
      • Selva E.
      microRNA regulation of Wnt signaling pathways in development and disease.
      ;
      • Tepekoy F.
      • Akkoyunlu G.
      • Demir R.
      The role of Wnt signaling members in the uterus and embryo during pre-implantation and implantation.
      ).
      Several miRNAs regulate the FoxO signalling pathway, controlling the expression of several genes, among which FOXO1 plays the most important role. In fact, it was demonstrated that mouse FoxO1, FoxO3 and FoxO4 proteins are regulated by the PI3K/Akt signalling pathway and are implicated in oocyte maturation and preimplantation embryo development (
      • Brosens J.J.
      • Wilson M.S.
      • Lam E.W.
      FOXO transcription factors: from cell fate decisions to regulation of human female reproduction.
      ;
      • Makker A.
      • Goel M.M.
      • Mahdi A.A.
      PI3K/PTEN/Akt and TSC/mTOR signaling pathways, ovarian dysfunction, and infertility: an update.
      ;
      • Kuscu N.
      • Celik-Ozenci C.
      FOXO1, FOXO3, AND FOXO4 are differently expressed during mouse oocyte maturation and preimplantation embryo development.
      ).
      It is known that follicular fluid represents a micro-environment that influences the oocyte competence and embryo development, thus, changing its composition could be considered a predictive factor of oocyte and embryo quality (
      • O'Gorman A.
      • Wallace M.
      • Cottell E.
      • Gibney M.J.
      • McAuliffe F.M.
      • Wingfield M.
      • Brennan L.
      Metabolic profiling of human follicular fluid identifies potential biomarkers of oocyte developmental competence.
      ). In addition, miRNAs are considered crucial players in the regulation of oocyte and embryo development (
      • Hossain M.M.
      • Salilew-Wondim D.
      • Schellander K.
      • Tesfaye D.
      The role of microRNAs in mammalian oocytes and embryos.
      ).
      To investigate the possible involvement of differentially expressed miRNAs in the determination of oocyte quality in more depth, we conducted a literature review of published data. Among the differentially expressed miRNAs, we found that miR-1303 is upregulated in follicular fluid of bad- quality oocytes (
      • Zhang D.
      • Lv J.
      • Tang R.
      • Feng Y.
      • Zhao Y.
      • Fei X.
      • Chian R.
      • Xie Q.
      Association of exosomal microRNAs in human ovarian follicular fluid with oocyte quality.
      ). It is involved in tumourigenesis and in the progression of several cancers. Its target genes are involved in the Hippo signalling pathway regulation, which is crucial for cell signalling in growth and development, regulating the activation of primordial follicles in mice (
      • Hu L.L.
      • Su T.
      • Luo R.C.
      • Zheng Y.H.
      • Huang J.
      • Zhong Z.S.
      • Nie J.
      • Zheng L.P.
      Hippo pathway functions as a downstream effector of AKT signaling to regulate the activation of primordial follicles in mice.
      ). It is also associated with ovarian ageing in mice (
      • Li J.
      • Zhou F.
      • Zheng T.
      • Pan Z.
      • Liang X.
      • Huang J.
      • Zheng L.
      • Zheng Y.
      Ovarian Germline Stem Cells (OGSCs) and the Hippo Signaling Pathway Association with Physiological and Pathological Ovarian Aging in Mice.
      ). miR-503-5p was shown to be directly involved in the formation and maintenance of corpus luteum (
      • Reza A.
      • Choi Y.J.
      • Han S.G.
      • Song H.
      • Park C.
      • Hong K.
      • Kim J.H.
      Roles of microRNAs in mammalian reproduction: from the commitment of germ cells to peri-implantation embryos.
      ).
      According to
      • Moreno J.M.
      • Nunez M.J.
      • Quinonero A.
      • Martinez S.
      • de la Orden M.
      • Simon C.
      • Pellicer A.
      • Diaz-Garcia C.
      • Dominguez F.
      Follicular fluid and mural granulosa cells microRNA profiles vary in in vitro fertilization patients depending on their age and oocyte maturation stage.
      , miR-574-5p is differentially expressed according to IVF patient age and oocyte maturation stage. This altered expression was observed in follicular fluid samples and granulosa cells obtained from each patient (
      • Moreno J.M.
      • Nunez M.J.
      • Quinonero A.
      • Martinez S.
      • de la Orden M.
      • Simon C.
      • Pellicer A.
      • Diaz-Garcia C.
      • Dominguez F.
      Follicular fluid and mural granulosa cells microRNA profiles vary in in vitro fertilization patients depending on their age and oocyte maturation stage.
      ). An altered expression in granulosa cells was also found for miR-423-5p. In particular, it is downregulated in the granulosa cells from patients with high ovarian response to exogenous gonadotrophins, suggesting that it may play an important role in the regulation of ovarian response (
      • Xie S.
      • Batnasan E.
      • Zhang Q.
      • Li Y.
      MicroRNA Expression is Altered in Granulosa Cells of Ovarian Hyperresponders.
      ;
      • Xie S.
      • Zhang Q.
      • Zhao J.
      • Hao J.
      • Fu J.
      • Li Y.
      MiR-423-5p may regulate ovarian response to ovulation induction via CSF1.
      ).
      Data show a general downregulation of let-7b-5p in follicular fluid and plasma from patients affected by endometriosis (
      • Papari E.
      • Noruzinia M.
      • Kashani L.
      • Foster W.G.
      Identification of candidate microRNA markers of endometriosis with the use of next-generation sequencing and quantitative real-time polymerase chain reaction.
      ) and in Hodgkin lymphoma cell lines (
      • Yuan Y.
      • Kluiver J.
      • Koerts J.
      • de Jong D.
      • Rutgers B.
      • Abdul Razak F.R.
      • Terpstra M.
      • Plaat B.E.
      • Nolte I.M.
      • Diepstra A.
      • Visser L.
      • Kok K.
      • van den Berg A.
      miR-24-3p Is Overexpressed in Hodgkin Lymphoma and Protects Hodgkin and Reed-Sternberg Cells from Apoptosis.
      ), and its possible association with oocyte quality. We previously detailed a strong downregulation of let-7b-5p in oocytes from women in advanced reproductive age (
      • Battaglia R.
      • Vento M.E.
      • Ragusa M.
      • Barbagallo D.
      • La Ferlita A.
      • Di Emidio G.
      • Borzi P.
      • Artini P.G.
      • Scollo P.
      • Tatone C.
      • Purrello M.
      • Di Pietro C.
      MicroRNAs Are Stored in Human MII Oocyte and Their Expression Profile Changes in Reproductive Aging.
      ).
      Let-7b-5p is also downregulated in follicular fluid from patients affected by polycystic ovary syndrome compared with women with a normal ovarian reserve and it seems to be associated with blastulation rate and, in general, it may reflect the abnormal folliculogenesis that characterizes polycystic ovary syndrome (
      • Scalici E.
      • Traver S.
      • Mullet T.
      • Molinari N.
      • Ferrieres A.
      • Brunet C.
      • Belloc S.
      • Hamamah S.
      Circulating microRNAs in follicular fluid, powerful tools to explore in vitro fertilization process.
      ).
      • Timofeeva A.V.
      • Chagovets V.V.
      • Drapkina Y.S.
      • Makarova N.P.
      • Kalinina E.A.
      • Sukhikh G.T.
      Cell-Free, Embryo-Specific sncRNA as a Molecular Biological Bridge between Patient Fertility and IVF Efficiency.
      demonstrated that the expression of let-7b-5 decreases in fair-quality blastocysts and is negatively correlated with both embryo development grade and number of metaphase II oocytes and two pronuclei cells.
      In summary, with the use of NanoString technology, we analysed 800 follicular fluid miRNAs and found that 13 miRNAs showed deregulation in women with Hodgkin lymphoma compared with young women not affected by Hodgkin lymphoma. Among them, five miRNAs had already been associated with oocyte quality and are part of a regulatory network in which let-7b-5p holds the central node of the network and shares mRNA target genes involved in follicle development and oocyte maturation with the other DE miRNAs. We also confirmed the significant downregulation of let-7b-5p in Hodgkin lymphoma follicular fluid samples compared with control patients in a larger cohort of follicular fluid samples. Although these data do not demonstrate that oocyte quality is lower in young women affected by Hodgkin lymphoma than in women of the same age, they suggest that critical conditions may affect the follicular microenvironment and influence oocyte competence. Moreover, Hodgkin lymphoma is a widespread tumour and, especially in the most advanced grades, it could also influence follicular fluid composition. Of course, it will take a few more years to obtain more information about the pregnancy rate and the number of live births from vitrified oocytes of Hodgkin lymphoma patients who had undergone protocols of fertility preservation. At present, this paper, representing the first study investigating miRNA profiles in follicular fluid from young women with Hodgkin lymphoma, could be the starting point for further molecular studies exploring the quality of the follicular microenvironment to improve counselling patients during the oncofertility protocol.
      A possible application could be to find biomarkers of oocyte competence in cumulus cells, granulosa cells or follicular fluid inside the single follicle, as described in a recent study (
      • Rooda I.
      • Hasan M.M.
      • Roos K.
      • Viil J.
      • Andronowska A.
      • Smolander O.P.
      • Jaakma Ü.
      • Salumets A.
      • Fazeli A.
      • Velthut-Meikas A.
      Cellular, Extracellular and Extracellular Vesicular miRNA Profiles of Pre-Ovulatory Follicles Indicate Signaling Disturbances in Polycystic Ovaries.
      ). In fact, we know that the retrieved oocytes from a single stimulation protocol do not necessarily have equivalent quality (
      • Di Pietro C.
      • Vento M.
      • Guglielmino M.R.
      • Borzi P.
      • Santonocito M.
      • Ragusa M.
      • Barbagallo D.
      • Duro L.R.
      • Majorana A.
      • De Palma A.
      • Garofalo M.R.
      • Minutolo E.
      • Scollo P.
      • Purrello M.
      Molecular profiling of human oocytes after vitrification strongly suggests that they are biologically comparable with freshly isolated gametes.
      ).
      The detection of molecular markers of oocyte quality could allow us to classify the retrieved oocytes before freezing. After cancer recovery, women have the chance to start a pregnancy. At this point, it could be possible to thaw and fertilize the most competent oocyte, previously classified, to improve the success of transfer and pregnancy rate. Moreover, the identification of altered pathways involved in follicle growth and oocyte maturation in women with Hodgkin lymphoma could help to design specific treatments that could improve reproductive potential.
      In conclusion, the deregulation of some miRNAs in follicular fluid from young women with Hodgkin lymphoma could affect the follicular microenvironment and consequently oocyte competence. Further studies are needed to improve our knowledge and help young women to become a mother after cancer.

      Acknowledgements

      The authors would like to thank the Scientific Bureau of the University of Catania for language support and Fondi di Ateneo 2020-2022, University of Catania, Open Access line.

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      Biography

      Cinzia Di Pietro is Associate Professor of Biology and Genetics at the University of Catania. Her group has studied cellular RNAs and exosomal microRNAs in ovarian follicles and in blastocoel fluid, associating their expression with oocyte and embryo quality, to understand their biological role in female gametogenesis and preimplantation embryo.
      Key message
      MicroRNAs are dysregulated in follicular fluid of women affected by Hodgkin Lymphoma, control pathways related to cancer and follicle development and could serve to predict the reproductive potential of patients with Hodgkin lymphoma.