Ileana Mateizel

Background: Approximately one-third of men having undergone gonadotoxic treatment in their childhood experience impaired testicular function for whom autologous transplantation of cryopreserved immature testicular tissue may represent the only opportunity to restore their fertility. Pre-clinical studies have demonstrated successful restoration of spermatogenesis following grafting of immature testicular tissue in various species, including non-human primates. In 2002, our institution pioneered with clinical testicular tissue banking for fertility preservation in boys and adolescents. Over time, this strategy has been increasingly implemented by numerous fertility centres worldwide for patients at high risk of treatment-induced sterility. Here, we report the first human case of autologous transplantation of frozen-thawed immature testicular tissue. Patient: In 2008, testicular tissue was cryopreserved from a pre-pubertal boy diagnosed with sickle cell disease. The procedure was performed after a three-year hydroxyurea treatment and prior to receiving conditioning therapy with busulfan and cyclophosphamide for haematopoietic stem cell transplantation. One testis was surgically removed, sectioned into small fragments, and cryopreserved. Histological analysis confirmed preserved tubular architecture and the presence of spermatogonia. During the period from 2022 to 2024, the patient consistently presented with azoospermia. In December 2024, at the time of transplantation, two abnormal sperm cells were detected after enzymatic digestion. Method: Eleven testicular tissue fragments (4–21 mm ³ ) were thawed and autologously grafted to four intra-testicular and four subcutaneous scrotal sites. Over a one-year follow-up period, graft survival, vascularization, hormone profiles, and semen parameters were monitored. One year after transplantation, all grafts were surgically retrieved. Results: Post-operative recovery was uneventful. No significant changes in endocrine or semen parameters were observed during follow-up. Whereas the intra-testicular grafts exhibited a compact parenchyma that was distinct from the looser surrounding adult parenchyma and remained readily identifiable as graft tissue, the scrotal grafts appeared more fibrotic. Enzymatic digestion of the grafts was required to recover spermatozoa, with one spermatozoon obtained from one of the four intra-testicular grafts. Histological evaluation revealed intact tubular architecture and maturation of somatic cells across all grafts. Spermatogonial stem cells, together with evidence of active spermatogenesis, were identified in two of the four intra-testicular grafts, whereas no germ cells were detected in the subcutaneous scrotal grafts. Conclusion: These findings demonstrate that human immature testicular tissue can survive long-term cryostorage, revascularize after transplantation and establish spermatogenesis in vivo . This study provides essential proof-of-concept for fertility restoration in individuals who banked testicular tissue before puberty. Funding: This study was supported by the Research Programme of FWO Vlaanderen (Research Foundation-Flanders; G0A6U25N) and VUB strategic research program (SRP89). Trial Registration: NCT05414045

Purpose To evaluate (1) HIV-1 viral load in pre- and post-processed testicular tissue samples from virally suppressed azoospermic HIV-positive men and (2) the reproductive outcome of the subsequent ICSI cycles. Methods This single-center case series reports on all virally suppressed HIV-positive men who underwent testicular biopsy at our center between 2015 and 2023. Whenever spermatozoa were present, samples underwent washing and viral load assessment in the first and last supernatant fractions. Cryopreservation of the last pellet was performed only if the final supernatant showed undetectable viral load. Embryological and clinical outcomes of subsequent ICSI cycles were evaluated. Results Six virally suppressed azoospermic HIV-positive men (mean age 43.1 ± 6.8 years) underwent seven biopsies, all yielding spermatozoa. All patients had frozen sperm available for ICSI with undetectable viral load following processing. Eight ICSI cycles were performed across six couples, with fertilization and embryo utilization rates of 57.9% (33/57) and 63.6% (21/33). Three fresh single embryo transfers (three couples) resulted in one monozygotic twin delivery. Twelve frozen embryo transfers (five couples) led to four live births and one elective termination. The cumulative live birth rate per couple was 50.0% (3/6). Conclusions We report cryopreservation of testicular tissue with undetectable viral load following sperm processing in virally suppressed HIV-positive men. Therapeutic application of these samples in ICSI-cycles results in successful clinical outcomes.

Purpose To identify the sperm preparation procedure that selects the best sperm population for medically assisted reproduction. Methods Prospective observational study comparing the effect of four different sperm selection procedures on various semen parameters. Unused raw semen after routine diagnostic analysis was split in four fractions and processed by four different methods: (1) density gradient centrifugation (DGC), (2) sperm wash (SW), (3) DGC followed by magnetic activated cell sorting (MACS), and (4) using a sperm separation device (SSD). Each fraction was analyzed for progressive motility, morphology, acrosome index (AI), and DNA fragmentation index (DFI). Results With DGC as standard of care in intraclass correlation coefficient analysis, only SSD was in strong disagreement regarding progressive motility and DFI [0.26, 95%CI (− 0.2, 0.58), and 0.17, 95%CI (− 0.19, 0.45), respectively]. When controlling for abstinence duration, DFI was significantly lower after both MACS and SSD compared to DGC [− 0.27%, 95%CI (− 0.47, − 0.06), p = 0.01, and − 0.6%, 95%CI (− 0.80, − 0.41), p < 0.001, respectively]. Further comparisons between SSD and MACS indicate significantly less apoptotic cells [Median (IQR) 4 (5), 95%CI (4.1, − 6.8) vs Median (IQR) 5 (8), 95%CI (4.9, − 9.2), p < 0.001, respectively] and dead cells [Median (IQR) 9.5 (23.3), 95%CI (13.2, − 22.4) vs Median (IQR) 22 (28), 95%CI (23.1, − 36.8), p < 0.001, respectively] in the SSD group. Conclusion The selection of a population of highly motile spermatozoa with less damaged DNA from unprocessed semen is ideally performed with SSD. Question remains whether this method improves the embryological outcomes in the IVF laboratory.

STUDY QUESTION: Is there an increase in the total number of metaphase II (MII) oocytes between a conventional ovarian stimulation (OS) and a double uninterrupted stimulation?SUMMARY ANSWER: There is no increase in the total number of MII oocytes when comparing one conventional OS to a continuous stimulation with double oocyte aspiration.WHAT IS KNOWN ALREADY: Based on the concept of multiple follicular waves, the combination of two stimulations in the same ovarian cycle has gained interest in patients with a low ovarian reserve. This so-called dual stimulation approach is usually characterized by a discontinuation of FSH administration for ∼5 days and appears to have a favourable impact on the number of retrieved oocytes without affecting the embryo quality or ploidy status. The outcomes of dual uninterrupted OS have not yet been studied.STUDY DESIGN, SIZE, DURATION: This was an open-label randomized controlled trial (RCT) with superiority design, performed in a single tertiary centre. Subjects were randomized with a 1:1 allocation into two groups between October 2019 and September 2021. All patients underwent a conventional stimulation with recombinant FSH. When two or more follicles of 17 mm were present, the final inclusion criterion was assessed; randomization occurred only in the presence of ≤9 follicles of ≥11 mm. In Group A, ovulation was triggered with hCG, and oocyte retrieval (OR) was performed 34–36 h later, followed by a fresh single or double embryo transfer (SET or DET) on Day 3/5. In Group B, ovulation was triggered with GnRH agonist, followed by another OS, without discontinuation of the FSH administration. In the presence of one or more follicles of ≥17 mm, the second stimulation was completed with hCG. A freeze-all strategy (Day 3/5) was applied for both retrievals, followed by transfer of one or two embryos in an artificially prepared frozen-thawed cycle. In the absence of one or more follicles of ≥17 mm after 13 additional days of stimulation, the second cycle was cancelled. All ORs were executed by a senior fertility specialist who was blinded for the first treatment, and all follicles >10 mm were aspirated, according to routine clinical practice. The primary outcome was the total number of MII oocytes. Patients were followed up until all embryos were transferred, or until live birth was achieved. Other secondary outcomes included the number of cumulus–oocyte complexes (COCs), the number of good quality embryos (Day 3/5), the ongoing pregnancy rate, and gonadotropin consumption.PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients between 25 and 40 years old, with an anti-Müllerian hormone level of ≤1.5 ng/ml, antral follicle count of ≤6, or ≤5 oocytes after a previous stimulation, were included. At the start, 70 patients were eligible for participation in the trial, of whom 48 patients fulfilled the final inclusion criterium and were randomized. After drop-out of two patients, 23 patients were randomized to a single round of OS (Group A), and 23 patients were randomized to two uninterrupted rounds of OS (Group B).MAIN RESULTS AND THE ROLE OF CHANCE: Baseline characteristics were similar between both groups. The cumulative number of COCs and MII oocytes after completion of the second OR was similar in Group A and Group B [5.3 ± 2.7 versus 5.3 ± 3.0 (P = 0.95); 4.1 ± 2.4 versus 4.3 ± 2.7 (P = 0.77)]. Likewise, a comparable number of excellent and good quality embryos was available on Day 3 (3.0 ± 2.0 versus 2.7 ± 2.0; P = 0.63). In Group B, the cancellation rate due to insufficient response to the second round of stimulation was 39.1% (9/23). When focusing on the first stimulation in both groups, there were no significant differences regarding basal FSH, gonadotropin consumption, and the number of preovulatory follicles. After the first OR, the mean number of COC and MII oocytes was significantly higher in Group A (who had hCG triggering), compared to Group B (who had GnRH agonist triggering) [5.3 ± 2.7 versus 3.3 ± 2.2; difference 95% CI (0.54 to 3.45), P = 0.004 and 4.1 ± 2.4 versus 3.0 ± 2.2; difference 95% CI (−0.15 to 2.6), P = 0.05, respectively]. Likewise, the number of excellent and good quality embryos on Day 3 was significantly higher (3.0 ± 2.0 versus 1.9 ± 1.7; P = 0.02) in Group A.LIMITATIONS, REASONS FOR CAUTION: This study was powered to demonstrate superiority for the number of MII oocytes after dual stimulation. Investigating the impact of dual stimulation on pregnancy rates would have required a larger sample size. Furthermore, the heterogeneity in embryo vitrification and transfer policies precluded a correct comparison of embryologic outcomes between both groups.WIDER IMPLICATIONS OF THE FINDINGS: This is the first RCT investigating the role of continuous stimulation with double aspiration in low responders. Our results show no statistically significant differences in the cumulative number of MII oocytes between one conventional stimulation with fresh ET and two consecutive stimulations with a freeze-only approach. Furthermore, the observed suboptimal oocyte yield after agonist ovulation triggering in low responders in the dual uninterrupted OS group is a reason for concern and further scrutiny, given that previous RCTs have shown similar outcomes in normal and high responders after hCG and GnRH agonist triggers.STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by a research grant from Organon. H.T. received honoraria for lectures and presentations from Abbott, Cooper Surgical, Gedeon-Richter, Cook, Goodlife, and Ferring. L.B. received fees for lectures from Merck & Organon and support for attending ESHRE 2023. M.D.V. reports fees for lectures from Ferring, Merck, Organon, IBSA, Gedeon Richter, and Cooper Surgical and support for attending ASRM 2023. S.M. received honoraria for lectures and...

STUDY QUESTION: What have we learnt after 10 years of electronic witnessing? SUMMARY ANSWER: When applied correctly, an electronic witnessing system can replace manual witnessing in the medically assisted reproduction lab to prevent sample mix-up. WHAT IS KNOWN ALREADY: Electronic witnessing systems have been implemented to improve the correct identification, processing, and traceability of biological materials. When non-matching samples are simultaneously present in a single workstation, a mismatch event is generated to prevent sample mix-up. STUDY DESIGN, SIZE, DURATION: This evaluation investigates the mismatch and administrator assign rate over a 10-year period (March 2011–December 2021) with the use of an electronic witnessing system. Radiofrequency identification tags and barcodes were used for patient and sample identification. Since 2011, IVF and ICSI cycles and frozen embryo transfer cycles (FET) were included; IUIs cycles were included since 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: The total number of tags and witnessing points were recorded. Witnessing points in a particular electronic witnessing system represent all the actions that have been performed from gamete collection through embryo production, to cryopreservation and transfer. Mismatches and administrator assigns were collected and stratified per procedure (sperm preparation, oocyte retrieval, IVF/ICSI, cleavage stage embryo or blastocyst embryo biopsy, vitrification and warming, embryo transfer, medium changeover, and IUI). Critical mismatches (such as mislabelling or non-matching samples within one work area) and critical administrator assigns (such as samples not identified by the electronic witnessing system and unconfirmed witnessing points) were selected. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 109 655 cycles were included: 53 023 IVF/ICSI, 36 347 FET, and 20 285 IUI cycles. The 724 096 used tags, led to a total of 849 650 witnessing points. The overall mismatch rate was 0.251% (2132/849 650) per witnessing point and 1.944% per cycle. In total, 144 critical mismatches occurred over the different procedures. The yearly mean critical mismatch rate was 0.017 ± 0.007% per witnessing point and 0.129 ± 0.052% per cycle. The overall administrator assign rate was 0.111% (940/849 650) per witnessing point and 0.857% per cycle, including 320 critical administrator assigns. The yearly mean critical administrator assign rate was 0.039 ± 0.010% per witnessing point and 0.301 ± 0.069% per cycle. Overall mismatch and administrator assign rates remained fairly stable during the evaluated time period. Sperm preparation and IVF/ICSI were the procedures most prone to critical mismatch and administrator assigns. LIMITATIONS, REASONS FOR CAUTION: The procedures and methods of integration of an electronic witnessing system may vary from one laboratory to another and result in differences in the potential risks related to sample identification. Individual embryos cannot (yet) be identified by such a system; this makes extra manual witnessing indispensable at certain critical steps where potential errors are not recorded. The electronic witnessing system still needs to be used in combination with manual labelling of both the bottom and lid of dishes and tubes to guarantee correct assignment in case of malfunction or incorrect use of radiofrequency identification tags. WIDER IMPLICATIONS OF THE FINDINGS: Electronic witnessing is considered to be the ultimate tool to safeguard correct identification of gametes and embryos. But this is only possible when used correctly, and proper training and attention of the staff is required. It may also induce new risks, i.e. blind witnessing of samples by the operator. STUDY FUNDING/COMPETING INTEREST(s): No funding was either sought or obtained for this study. J.S. presents webinars on RIW for CooperSurgical. The remaining authors have nothing to declare. TRIAL REGISTRATION NUMBER: N/A.

To investigate whether treatment with commercially available ready-to-use A23187 ionophore (GM508-CultActive) improves embryo development outcome in patients with a history of embryo developmental problems. This is a uni-center prospective study in which sibling oocytes of patients with embryos of poor quality on day 5 in the previous cycle were treated or not with CultActive. Two hundred forty-seven metaphase II (MII) oocytes from 19 cycles performed between 2016 and 2019 were included in the study. After ICSI, the sibling oocytes were assigned to the treatment group or to the control group, following an electronically generated randomization list. A number of 122 MII were treated with CultActive and 125 MII had no treatment and were assigned to the control group. No difference in fertilization rate (p = 0.255) or in the capacity of embryos to reach good quality on day 5 (p = 0.197) was observed between the two groups. The utilization rates defined as the number of embryos transferred or cryopreserved per mature oocyte (p = 0.438) or per fertilized oocytes (p = 0.299) were not significantly different between the treated group and the control group. The results of the current study do not support the use of CultActive in cases with embryo developmental problems.

Calcium ionophore treatment is being used in assisted reproductive technology (ART) for cases with previous low fertilization rate or total absence of fertilization after insemination by intracytoplasmic sperm injection or when a specific indication such as globozoospermia is present. As this technique is more invasive and differs from the physiological process of fertilization, a thorough investigation of the health of the children born following this procedure is required. We intent to report the medical outcome of all children conceived following calcium ionophore treatment in our IVF center.

STUDY QUESTION Does the manipulation of gametes or embryos during ARTs increase the risk for monozygotic twinning (MZT)? SUMMARY ANSWER Frozen embryo transfer (ET) is associated with a lower MZT rate, while blastocyst culture is associated with an increased risk of monozygotic pregnancy. WHAT IS KNOWN ALREADY Monozygotic twins have a higher risk for perinatal complications. Although an increased incidence of monozygotic pregnancies after ART has been previously reported, data regarding the possible impact of different laboratory procedures are conflicting. STUDY DESIGN, SIZE, DURATION All clinical pregnancies after single ET carried out in our centre between 2004 and 2013 (n = 6096) were retrospectively analysed for the incidence of MZT. The effect of different laboratory procedures on the incidence of MZT was evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS The following ART risk factors were assessed: maternal age, type of ET (fresh versus frozen), zona pellucida (ZP) manipulation (specifically, ICSI, embryo biopsy and assisted hatching), use of donor oocytes, embryo stage at time of ET (cleavage, compaction, early or advanced blastocyst) and culture media. MAIN RESULTS AND THE ROLE OF CHANCE The overall MZT rate was 2.2% (136/6096). Frozen ET was associated with a significant reduction in MZT incidence (adjusted odds ratio (aOR) 0.48, 95% CI 0.29–0.80), while blastocyst transfer (early or advanced blastocyst) was associated with a significant increase in MZT risk (aOR 2.70, 95% CI 1.36–5.34; aOR 2.05, 95% CI 1.29–3.26, respectively). No significant differences were found between the MZT and singleton (non-MZT) groups regarding maternal age, the use of different ZP manipulation techniques, not type of culture media used. LIMITATION, REASONS FOR CAUTION This study is limited by its retrospective nature and the fact that monozygosity was not confirmed by genetic testing. Furthermore, since monozygotic pregnancy is a rare event, other ART parameters that may influence its incidence could not be assessed during our analysis. WIDER IMPLICATION OF THE FINDINGS Our findings warrant future studies designed to investigate the association between specific ART procedures and MZT, namely the potential risk of blastocyst transfer to increase MZT. STUDY FUNDING/COMPETING INTEREST(S) No external funding was used for this study. There are no conflicts of interest.