ABSTRACT: Cryopreservation process deregulates the expression of pathways important for fertility in human spermatozoa Study design, size, duration (max. 75 words): Semen samples were obtained from 20 normospermic men from April to May 2022. Each sample was divided in two aliquots. From one aliquot total RNA was immediately extracted. The second aliquot was slowly cryopreserved and after a week of storage in liquid nitrogen total RNA was extracted. A total of 13 paired RNA samples passed quality control and were analyzed after randomization into 4 pools, each of 6 patients. Participants/materials, setting, methods (max. 75 words): The men who participated in this study had a median age of 35.0 years (range: 29.0-46.0). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The paired Significance Analysis of Microarray (SAM) was performed using the limma, hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance (max. 200 words): The expression profiles of cryopreserved sperm significantly differed from those of fresh sperm in 219 down-regulated and 28 up-regulated unique transcripts. The gene ontology analysis disclosed that cryopreservation downregulates genes involved in sperm motility by the mitochondria function (CABS1, SPATA19) and the correct organization of the sperm midpiece and flagellum cytoskeleton (ACTL7A, AKAP4, C9ORF24, CAPZB, CCIN, IQCG, ODF2, SPATA6, SPATA6L, TBC1D21), in fertilization (ACTL7A, AKAP4, PLCZ1, PRSS37, TUBGCP3), early embryo development (AGT, CLMN, DCC, DHRS3, EGFLAM, FAM20A, FREM1, GPI, HEMGN, KRTDAP, IFT172, MICAL2, MLF1, NF2, PGRMC2, PHACTR1, POPDC3, RO60, ROBO1, RORA, SGCA, SPRR2D, TCF4, TNC, TNNI3), oxidant detoxification (TXNDC29) and DNA repair (BRIP1, ERCC6, TRIP12), calcium ion binding and homeostasis (AMY1C, ANO1, ANO2, ASGR1, CABS1, CPNE9, FREM1, KCNIP2, ITPR3, PKD2L1, PLCZ1, SELENOK, SYN3, TNNI3). Upregulated genes were enriched in pathways related to the negative regulation of DNA damage response (ALOX15B, CD74, RPL10, SNAI1, THBS1), cellular response to calcium ion (ALOX15B, FOSB, HLA-DQB1, S100A4, THBS1), regulation of transcription (CD74, FOSB, HLA-DRB1, MAMLD1, RASD1, RPL10, SNAI1, TCF15, TLE1), protein stabilization (CD74), protein ubiquitination (BAG1, DCAF6, ERCC6, HERPUD2, KLHL7, MARCHF8, NF2, PSMA6, RNF133, SERGEF, TRIP12, UBE2DNL, UBL3, UBQLN3, ZNRF3) and ubiquitin protein ligase binding (GPI, HSPA1L, PRKAR2A, STX8). The storage time of cryopreserved human spermatozoa does not affect pathways involved in fertility. Study design, size, duration This study included 24 normospermic men after their written informed consent, of whom 13 had cryostored semen for a short-time (1 week) and 11 had cryostored semen for a long-time (median 9 years, range 7-10). RNA was extracted from each frozen-thawed sperm sample and randomized into pools (4 pools for short-time samples and 3 pools for long-time samples), each of 5-6 patients. Wide-transcriptome comparison of short- and long-time pools were analyzed. Participants/materials, setting, methods The study population had a median age of 34.5 years (range: 22-46), without significant difference between the two groups (p-value = 0.147). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The unpaired Significance Analysis of Microarray (SAM) was performed using the limma, hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance Comparison of gene expression profiles between thawed sperm after a short or long storage period in liquid nitrogen identified 5 unique transcripts significantly upregulated in the latter group (DCP1A, FAM217A, HUWE1, MSI2, PCSKIN). Functional annotation enrichment has shown that the dysregulated transcripts are involved in methylation and protein binding. In particular, HUWE1 encodes an E3 ubiquitin protein ligase required for protein ubiquitination. The protein encoded by DCP1A is a decapping enzyme, necessary for the degradation of mRNAs. The RNA binding protein MSI2 regulates the expression of target mRNAs at the translation level. Interestingly, HUWE1 serves as a central node in cellular stress responses, cell growth and death, signal transduction, etc. During early embryonic development, siRNA-mediated knockdown of HUWE1 induced apoptotic cell death and inhibited normal embryonic development. Consistent with this finding, decreased HUWE1 staining in human embryos was found to be associated with poor embryo quality, suggesting that HUWE1 plays important roles in promoting embryonic development. DCP1A is also essential for embryonic development. Overall, no critical pathways involved in fertility are dysregulated during storage of sperm in liquid nitrogen and upregulation of genes important for early embryonic development may compensate for sperm cryoinjury.
