Diminished ovarian reserve (DOR) is associated with a reduced quantity and quality of the retrieved oocytes, usually leading to poor reproductive outcomes which remain a great challenge for assisted reproduction technology (ART). Women with DOR often have to seek for oocyte donation, precluding genetically related offspring. Germline nuclear transfer (NT) is a novel technology in ART that involves the transfer of the nuclear genome from an affected oocyte/zygote of the patient to the cytoplast of an enucleated donor oocyte/zygote. Therefore, it offers opportunities for the generation of genetically related embryos. Currently, although NT is clinically applied only in women with serious mitochondrial DNA disorders, this technology has also been proposed to overcome certain forms of female infertility, such as advanced maternal age and embryo developmental arrest. In this review, we are proposing the NT technology as a future treatment option for DOR patients. Strikingly, the application of different NT strategies will result in an increase of the total number of available reconstituted embryos for DOR patients.
Retroelements are genetic mobile elements, expressed during male and female gamete differentiation. Retrotransposons are normally regulated by the methylation machinery, chromatin modifications, non-coding RNAs, and transcription factors, while retrotransposition control is of vital importance in cellular proliferation and differentiation process. Retrotransposition requires a transcription step, by a cellular RNA polymerase, followed by reverse transcription of an RNA intermediate to cDNA and its integration into a new genomic locus. Long interspersed elements (LINEs), human endogenous retroviruses (HERVs), short interspersed elements (SINEs) and SINE-VNTR-Alu elements (SVAs) constitute about half of the human genome, play a crucial role in genome organization, structure and function and interfere with several biological procedures. In this mini review, we discuss recent data regarding retroelement expression (LINE-1, HERVK-10, SVA and VL30) and retrotransposition events in mammalian oocytes and spermatozoa, as well as the importance of their impact on human and mouse preimplantation embryo development.Retroelements are genetic mobile elements that constitute 43% of the human and 37% of the mouse genome (1). They have been cited as "junk DNA", but recent evidence support that they play a crucial role in genome organization, structure, and function during evolution, as they can relocate themselves from one locus to another using an RNA intermediate (2, 3). The retrotransposition process requires a transcription step, by a cellular RNA polymerase, followed by reverse transcription of an RNA intermediate to cDNA and its integration into a new genomic locus (4).Retroelements are categorized, according to structural and functional criteria, in Long terminal repeats (LTR) and non-LTR as well as in autonomous and non-autonomous. LTR retrotransposons include Human endogenous retroviruses (HERVs), whereas non-LTR retrotransposons contain mainly the classes of Long interspersed elements (LINEs) and Short interspersed elements (SINEs) (5, 6) (Figure 1).LINEs and HERVs are autonomous retroelements, as they are able to encode reverse transcriptase and proteins necessary for their retrotransposition (7, 8). On the contrary, SVA (SINE-VNTR-Alu) and Alu elements, as nonautonomous, use the LINE-1 protein machinery for their mobilization and relocation (9-11). LINE-1 retroelements move between loci through the target primed reverse transcription (TPRT) mechanism. LINE-1 retrotransposition starts with the binding of RNA polymerase on the promoter of the 5' UTR region and the production of LINE-1 mRNA in the cell nucleus. LINE-1, mRNA moves to the cytoplasm where the ORF1 and ORF2 proteins are expressed. LINE-1 mRNA, ORF1p, and ORF2p are reinserted into the nucleus as a rivonucleoprotein (RNP) complex. ORF2 acts as an endonuclease that breaks the target DNA in the new site of retrotransposons insertion, producing a single stranded edge.
STUDY QUESTION Can spindle transfer (ST) overcome inferior embryonic development of in vitro matured ovarian tissue oocytes (OTO-IVM) originating from testosterone-treated transgender men? SUMMARY ANSWER ST shows some potential to overcome the embryo developmental arrest observed in OTO-IVM oocytes from transgender men. WHAT IS KNOWN ALREADY OTO-IVM is being applied as a complementary approach to increase the number of oocytes/embryos available for fertility preservation during ovarian tissue cryopreservation in cancer patients. OTO-IVM has also been proposed for transgender men, although the potential of their oocytes remains poorly investigated. Currently, only one study has examined the ability of OTO-IVM oocytes originating from transgender men to support embryo development, and that study has shown that they exhibit poor potential. STUDY DESIGN, SIZE, DURATION Both ovaries from 18 transgender men undergoing oophorectomy were collected for the purposes of this study, from November 2020 to September 2022. The patients did not wish to cryopreserve their tissue for fertility preservation and donated their ovaries for research. All patients were having testosterone treatment at the time of oophorectomy and some of them were also having menses inhibition treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS Sibling ovaries were collected in either cold or warm medium, to identify the most optimal collection temperature. Cumulus oocyte complexes (COCs) from each condition were isolated from the ovarian tissue and matured in vitro for 48 h. The quality of OTO-IVM oocytes was assessed by calcium pattern releasing ability, embryo developmental competence following ICSI, and staining for mitochondrial membrane potential. In vitro matured metaphase I (MI) oocytes, germinal vesicle (GV) oocytes, and in vivo matured oocytes with aggregates of smooth endoplasmic reticulum (SERa) were donated from ovarian stimulated women undergoing infertility treatment and these served as Control oocytes for the study groups. ST was applied to overcome poor oocyte quality. Specifically, enucleated mature Control oocytes served as cytoplasmic recipients of the OTO-IVM spindles from the transgender men. Embryos derived from the different groups were scored and analysed by shallow whole genome sequencing for copy number variations (CNVs). MAIN RESULTS AND THE ROLE OF CHANCE In total, 331 COCs were collected in the cold condition (OTO-Cold) and 282 were collected in the warm condition (OTO-Warm) from transgender men. The maturation rate was close to 54% for OTO-Cold and 57% for OTO-Warm oocytes. Control oocytes showed a calcium releasing ability of 2.30 AU (n = 39), significantly higher than OTO-Cold (1.47 AU, P = 0.046) oocytes (n = 33) and OTO-Warm (1.03 AU, P = 0.036) oocytes (n = 31); both values of calcium release were similar between the two collection temperatures. Mitochondrial membrane potential did not reveal major differences between Control, OTO-Warm, and OTO-Cold oocytes (P = 0.417). Following ICSI, 59/70 (84.2%) of Control oocytes were fertilized, which was significantly higher compared to 19/47 (40.4%) of OTO-Cold (P < 0.01) and 24/48 (50%) of OTO-Warm oocytes (P < 0.01). In total, 15/59 (25.4%) blastocysts were formed on Day 5 in the Control group, significantly higher than 0/19 (0%) from the OTO-Cold (P = 0.014) and 1/24 (4.1%) in OTO-Warm oocytes (P = 0.026). Application of ST rescued the poor embryo development, by increasing the Day 5 blastocyst rate from 0% (0/19) to 20.6% (6/29) (P = 0.034), similar to that in the ICSI-Control group (25.4%, 15/59). A normal genetic profile was observed in 72.7% (8/11) of OTO-Cold, 72.7% (8/11) of OTO-Warm and 64.7% (11/17) of Control Day 3–Day 5 embryos. After ST was applied for OTO-IVM oocytes, 41.1% (7/17) of the embryos displayed normal genetic patterns, compared to 57.1% (4/7) among ST-Control Day 3–Day 5 embryos. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Due to the limited access to human oocytes and ovarian tissue, our results should be interpreted with some caution, as only a limited number of human oocytes and embryos could be investigated. WIDER IMPLICATIONS OF THE FINDINGS The results of this study, clearly indicate that OTO-IVM oocytes originating from transgender patients are of inferior quality, which questions their use for fertility preservation. The poor quality is likely to be related to cytoplasmic factors, supported by the increased blastocyst numbers following application of ST. Future research on OTO-IVM from transgender men should focus on the cytoplasmic content of oocytes or supplementation of media with factors that promote cytoplasmic maturation. A more detailed study on the effect of the length of testosterone treatment is also currently missing for more concrete guidelines and guidance on the fertility options of transgender men. Furthermore, our study suggests a potentially beneficial role of experimental ST in overcoming poor embryo development related to cytoplasmic quality. STUDY FUNDING/COMPETING INTEREST(S) A.C. is a holder of FWO grants (1S80220N and 1S80222N). A.B. is a holder of an FWO grant (1298722N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) and 2018000504 (GOA030-18 BOF) funding. B.H. has additional grants from FWO-Vlaanderen (Flemish Fund for Scientific Research, G051516N and G1507816N) and Ghent University Special Research Fund (Bijzonder Onderzoeksfonds, BOF funding (BOF/STA/202109/005)), and has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER N/A.
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