PurposeTo describe the transgender population attending our clinics, the treatments of choice, and their clinical outcomes. To evaluate whether gender-affirming hormone therapy (GAHT) with testosterone in transmen affected ovarian stimulation, and the embryological and clinical outcomes.MethodMulticenter retrospective observational study analyzing transgender patients attending fertility clinics. We analyzed demographic, embryological, and clinical information regarding fertility preservation (FP) in transmen and transwomen, intrauterine insemination, intracytoplasmic sperm injection, third-party oocyte donation, and reception of oocytes from partner (ROPA) cycles among transmen or their partners. ROPA and oocyte-FP cycles were divided according to whether the patient was receiving testosterone therapy before stimulation (Tpos) or not (Tneg). We also described a preimplantation genetic testing for aneuploidies ROPA cycle undergone by a Tpos transman.ResultsTransgender individuals have increasingly undergone ART procedures over the years with a trend towards treatments in which transmen are biologically involved. Nonetheless, cycles analyzed were predominantly performed in their cis partners using sperm banks, with the choice depending on the partner's age. Embryological and clinical outcomes were comparable to the general population. The sample size of transwomen seeking FP was limited, showing abnormal sperm parameters that may have been influenced by estrogen and antiandrogen therapy. Tpos or Tneg cycles did not significantly differ in the analyzed stimulation, embryological, or clinical parameters.ConclusionsThe preferred treatment may be influenced by the occurrence of GAHT or gender affirming surgery (GAS) and the desire for biological involvement. Testosterone therapy before ovarian stimulation does not seem to alter embryological or clinical outcomes.

In vitro maturation (IVM) of immature oocytes has been explored for research and clinical purposes since the dawn of assisted reproductive technologies. Oocyte maturation is a highly specific process, on the basis of complex mutual relationships between the germ and somatic cell compartments. The complexity of this relationship has made the quest for achieving oocyte maturation in vitro arduous. In its classical form, in which intact cumulus-enclosed oocytes are collected after very mild or no ovarian stimulation, oocyte IVM is nonexperimental and has been proposed as a more friendly treatment for patients with polycystic ovary or polycystic ovary syndrome. By contrast, rescue IVM (r-IVM), which is the maturation in vitro of immature oocytes collected in standard ovarian stimulation cycles, is an experimental procedure, proposed to mitigate the impact of low oocyte maturation rates in certain patients. Achieving effective r-IVM has turned out to be an even more daunting task because oocytes are cultured only after cumulus cell removal and, therefore, without the crucial somatic metabolic and regulative support. Immature oocyte arrested at the germinal vesicle or metaphase I stage require different management for their maturation in vitro and exhibit different developmental and chromosomal competence. Therefore, their possible use for treatment suggests a dedicated approach. Overall, r-IVM has limited clinical efficacy due to suboptimal maturation and developmental competence of immature oocytes. This raises a cost/benefit question: that is, the definition of appropriate clinical indications. Rescue IVM is probably irrelevant to treatment cycles in which the absolute number of mature oocytes is high. Conversely, specific poor prognosis cases, involving low maturation rates, low oocyte yield, and/or low oocyte quality, could benefit from the contribution of even a single embryo generated from an in vitro matured oocyte. Future progress in this field will depend on our ability to mimic in vitro the support provided by cumulus cells to oocyte nuclear and cytoplasmic maturation.

This study aimed to identify demographic and clinical factors associated with low maturation rates and to investigate if the rate of immature oocytes impacts the outcomes of mature sibling oocytes. Women undergoing their first IVF-ICSI cycle between 2018 and 2022 at a fertility clinic were included. Cycles were classified into five groups according to the proportion of Metaphase II stage oocytes (MII): Null (0

Is there any difference in euploidy rates between in vitro fertilization (IVF) cycles triggered with GnRHa, hCG or dual trigger? There is no difference in the euploidy rate in preimplantation genetic testing for aneuploidy (PGT-A) cycles according to the trigger method used. The use of PGT-A has shown that the success of meiosis in the IVF laboratory is inversely correlated with maternal age. However, other uninvestigated factors involved in the IVF process may interfere with normal oocyte physiology. Since the final maturation obtained after ovarian stimulation depends on the use of drugs to promote the resumption of meiosis, it is worth investigating whether the triggering method could impact on oocyte and embryo euploidy. This study aims is to evaluate differences in embryo euploidy rates between PGT-A cycles triggered with GnRHa, hCG or dual trigger (GnRHa + hCG). This is a single-center retrospective study of 934 patients who underwent 1085 PGT-A cycles by Next-Generation Sequencing (NGS) from January 2017 to December 2023, using one of three possible trigger methods to induce final oocyte maturation: GnRHa, hCG, or dual trigger. The couples included in the study had normal karyotypes and no severe male factor. Data on the number of oocytes retrieved, ratio of metaphase II (MII) oocytes and fertilization rate, blastocyst development and embryo euploidy were assessed and compared between the GnRHa, hCG and dual trigger treatments. A secondary analysis was performed to evaluate data stratified by patient age. A total of 1085 PGT-A cycles involving 934 patients (38.07 ± 3.01 years) and 2803 biopsied blastocysts were included in the analysis. Dual trigger was used in 726 (77.73%) cycles, GnRHa in 287 (26,4%) cycles and hCG in 72 (7.71%) cycles. The number of oocytes retrieved was significantly higher in the GnRHa group (GnRHa: 18.72 ± 8.59; hCG: 9.88 ± 5.64; dual trigger: 10.58 ± 5.65; p < 0.001) with similar maturation rate (GnRHa: 77.46%; hCG: 74.58%; dual trigger: 76.03%; p = 0.789), fertilization rate (GnRHa: 78.12%; hCG: 76.37%; dual trigger: 79.12%; p = 0.168), blastulation rate (GnRHa: 61.03%; hCG: 58.32%; dual trigger: 58.73%; p = 0,629), high-quality blastocyst rate (GnRHa: 27,88%; hCG: 33,67%; dual trigger: 27,88%; p = 0.543) and embryo euploidy per biopsied embryo (GnRHa: 42.48%; hCG:45.49%; dual trigger:46.98%; p = 0.112). Analysis of the data stratified by age showed similar rates of euploidy rates with the three trigger methods in women younger than 41 years (<35 years: GnRHa 66.50%; hCG 66.67%; dual trigger 56.97% p = 0.290; 35-37 years: GnRHa 49.45% hCG 51.04%; dual trigger 50.23%; p = 0.988; 38-40 years: GnRHa 40.93%; hCG 41.27%; dual trigger 39.13% p = 0.257). However, in patients ≥41 years old the euploidy rate was statistically higher in the GnRHa group (GnRH 31.51%; hCG 18.75%; dual trigger 16.89%; p = 0.032). The main limitations of the study are: its retrospective design, the possibility that the trigger method was chosen based on the ovarian response and the limited number of patients in both the hCG trigger and women ≥ 41 years groups. Our results suggest that triggering with GnRHa, hCG or dual trigger leads to comparable euploidy rates. Triggering with GnRHa should be recommended in older patients since it has been associated with a higher rate of embryonic euploidy. Not Applicable

Abstract Study question What is the cause of loss of heterozygosity, frequently observed in embryos subjected to CRISPR-Cas9, an interhomolog-homologous recombination repair (IH-HR) or deficiency in DNA repair? Summary answer Vast majority of LOH observed in human zygotes subjected to CRISPR-based editing is explained by deficiency in DNA repair prior to genome activation, not IH-HR. What is known already Genome editing (GE) at the preimplantation stage, intended to correct disease-causing mutations, is controversial due to safety and ethical concerns. It has been suggested that a high frequency of LOH seen in zygotes subjected to GE could be explained by resolution of the double-stranded DNA breaks (DSBs) created using CRISPR-Cas9 by interhomolog-homologous recombination repair (IH-HR), a process with potential to correct deleterious heterozygous mutations. However, such hypotheses are controversial and it is unclear whether embryos at this stage development can utilise IH-HR. CRISPR-Cas9 targeted sites are difficult to interrogate with traditional methods, risking a misinterpretation of editing outcomes. Study design, size, duration We hypothesised that the frequently observed LOH affecting the CRISPR-targeted sites in human zygotes is caused by the deficiency in DNA repair at this developmental stage, resulting in complex genotypes which traditional molecular methods have failed to detect, and not IH-HR which uses the second un-cleaved parental allele as a template for repair. DSBs were induced at three genomic sites in embryos microinjected at the time of fertilisation (n = 27) in this IRB-approved study. Participants/materials, setting, methods Research embryos were cultured for 60 hours, then disaggregated. Individual cells underwent an array of genetic tests, including NGS, allowing determination of the repair pathway utilised and revealing any cytogenetic abnormalities. A novel state-of-the-art analytical workflow was developed to interrogate 25kb around CRISPR cleavage sites. This approach provided a means of detecting complex genomic rearrangements as well as unresolved DNA damage, which would appear as LOH when employing traditional PCR-based approaches for assessment. Main results and the role of chance Induction of DSBs using CRISPR-Cas9 was 100% efficient when applied at the time of fertilisation. LOH was confirmed to be common at targeted sites, affecting 69/216 (32%) loci examined in 108 blastomeres. Remarkably, the great majority of LOH events (>90%) were a direct consequence of inappropriate/failed DNA repair (unresolved DNA breaks or complex genomic rearrangements). Strikingly, not a single instance of IH-HR was observed when CRISPR reagents were introduced at fertilisation. This may be explained by the physical separation of the two parental genomes in distinct pronuclei at that time. Allele-drop out (ADO), a technical problem, accounted for <10% of all LOH events. In total, 46% of DSBs failed to undergo appropriate repair, resulting in segmental aneuploidy (21%), large-scale deletions (10%), inversions/translocations (10%), or loss of the targeted chromosome (5%). 45% of DSBs were resolved using error-prone non-homologous end joining (NHEJ), while only 9% utilised the homology directed repair (HDR) pathway required for correction of mutations. Application of CRISPR-Cas9 to embryos on day-3 of development (after embryonic genome activation - EGA) was less efficient, only producing a DSB in 6/37 embryos (16%). However, the mode of repair differed markedly, 100% employing HDR (p = 0.0001). In 4 cases, this involved IH-HR. Limitations, reasons for caution This data provides strong evidence that LOH recorded in earlier studies was incorrectly attributed to IH-HR and was actually due to abnormal/failed repair. CRISPR appears more effective after EGA, with efficient DNA repair dominated by desirable HDR. However, a larger number of embryos is required to corroborate this finding. Wider implications of the findings The use of CRISPR-Cas9 at the time of fertilisation is frequently associated with problematic, unintended outcomes, rendering the technique clinically inapplicable in the current format. Careful study design which allows determination of all possible editing outcomes is essential and advances our understanding of the underlying mechanisms of DNA repair. Trial registration number not applicable

Both spontaneously conceived pregnancies and those achieved using assisted reproduction decline with advancing maternal age. In this study, we tested if rapamycin and/or cumulus cells (CCs) from young donors could improve oocyte maturation and euploidy rates of germinal vesicle (GV) stage oocytes obtained from older women of reproductive age. A total of 498 GVs from 201 women >38 years (40.6 ± 1.8, mean ± SD) were included. GVs were randomly assigned into five groups for rescue IVM: control (with no CCs and no rapamycin); with autologous CCs; with autologous CCs and rapamycin; with CCs from young women (<35 years); and with CCs from young women and rapamycin. After 24 h of culture, the first polar body (PB) was biopsied in metaphase II oocytes, and the cytogenetic constitution was assessed using next-generation sequencing for both oocytes and PBs. Comparable maturation rates were found (56.2%, 60.0%, 46.5%, 51.7%, and 48.5% for groups 1-5, respectively; P = 0.30). Similarly, comparable euploidy rates were observed in the five groups (41.5%, 37.8%, 47.2%, 43.6%, and 47.8% for Groups 1-5, respectively; P = 0.87). Our findings indicate that rescue IVM is effective for obtaining mature euploid oocytes in older women of reproductive age, and that incubation with rapamycin or CCs obtained from young donors does not improve the maturation or euploidy rate.

Research question: Does rescue in -vitro maturation (IVM) in the presence or absence of cumulus cells, affect the progress of meiosis I, compared with oocytes that mature in vivo? Design: This prospective study was conducted in a university-affiliated fertility centre. Ninety-five young oocyte donors (mean age 25.57 4.47) with a normal karyotype and no known fertility problems were included. A total of 390 oocytes (116 mature metaphase II [MII] and 274 immature oocytes) were analysed. The immature oocytes underwent rescue IVM in the presence of cumulus cells (CC; IVM+CC; n =137) or without them (IVM-CC; n =137), and IVM rate was calculated. Chromosome copy number analysis using next-generation sequencing (NGS) was performed on all rescue IVM oocytes reaching MII as well as those that were mature at the time of initial denudation (in-vivo-matured oocytes [IVO]). Results: Maturation rates were similar in IVM+CC and IVM-CC oocytes (62.8 versus 71.5%, P = 0.16). Conclusive cytogenetic results were obtained from 65 MII oocytes from the IVM+CC group, 87 from the IVM-CC group, and 99 from the IVO group. Oocyte euploidy rates for the three groups were similar, at 75.4%, 83.9% and 80.8%, respectively (P = 0.42). Conclusions: The results suggest that culture of germinal vesicle and metaphase I oocytes in the presence of cumulus cells does not improve rates of IVM. In general, the process of rescue IVM does not appear to alter the frequency of oocytes with a normal chromosome copy number.

In a preimplantation genetic testing for aneuploidy (PGT-A) cycle, does the blastocyst quality before biopsy, or the day of biopsy, or the embryo hatching status have an impact on either euploidy or the rate of embryo survival after freezing? This was a retrospective study including 6130 biopsied blastocysts coming from 1849 PGT-A cycles performed in our center (2016–2022). Embryos were categorized according to the inner cell mass and trophectoderm quality, using Gardner’s scoring (excellent: AA; good: AB, BA, BB; poor: AC, CA, BC, CB, CC); the day of biopsy (5 or 6); and their hatching status (fully hatched blastocysts [FHB] or non-fully hatched blastocysts [nFHB]). The independent relationship between each group and both euploidy and survival rate was assessed. Excellent-quality embryos were more euploid than both good- and poor-quality embryos (52.69

Abstract Study question Are fragmentation levels (single-double stranded DNA breaks) or aneuploidy frequencies in sperm increased in patients with testicular cancer who recovered spermatogenesis after chemotherapy (CT)? Summary answer Similar levels of sperm DNA fragmentation and sperm aneuploidy frequency are found in men with testicular cancer pre- and post-chemotherapy. What is known already Testicular cancer is the most frequent solid tumor among young men. Therefore, the restoration of fertility and achievement of fatherhood in survivors have become important concerns. The integrity of genetic material in sperm is crucial since some paternal genes are essential for early embryo development. Although previous studies analyzed the effect of CT on sperm DNA integrity, they did not differentiate single vs. double-stranded DNA breaks. Few data are available on aneuploidy in sperm from testicular cancer survivors, so it remains to be elucidated whether cancer treatments may lead to an increased risk of aneuploidy syndromes in offspring. Study design, size, duration Prospective multicenter study including 31 males with a history of testicular cancer who requested semen cryopreservation between 2007-2021. All patients included underwent CT and recovered spermatogenesis once treatment ended. Post-CT samples were collected and seminal parameters, single-double stranded DNA breaks and the proportion of sperm aneuploidies were compared with one aliquot of the initial ejaculate. The project was approved by an Institutional Review Board and written informed consent for participation was obtained from each subject. Participants/materials, setting, methods Patient´s age prior CT was 31.23yrs (IC 95%: 29.29-33.17). Mean interval time between 1st and 2nd samples was 4.53yrs (IC 95%: 3.42-5.64). TUNEL quantified overall sperm DNA fragmentation by flow cytometry analyzing at least 20,000 cells, while γH2AX staining was applied for double DNA fragmentation assessment. Sperm aneuploidies were calculated by using fluorescence in situ hybridization (FISH) probes for chromosomes 13, 18, 21, X, Y. A paired t-test was used to compare variables pre- and post-CT. Main results and the role of chance Similar results were found when sperm quality parameters were compared. Mean sperm concentration pre-CT was 33.03M/mL (IC 95%: 23.98-42.07) and 36.41 M/mL (IC 95%: 27.85-44.96) in post-CT ejaculates (N.S.). A comparable rate of motile spermatozoa was observed in pre-CT vs. post-CT samples [(43.81% (IC 95%: 38.42-49.19) vs. 44.73% (IC 95%: 38.95-50.51), N.S.]. Regarding TUNEL assay results, the sperm DNA fragmentation rate was 21.65% (IC 95%: 17.66-25.63) in pre-CT samples and 23.00% (IC 95%: 19.90-26.61) in post-CT samples (N.S.). Furthermore, similar results were obtained when double-stranded fragmentation levels (H2AX) were compared between pre and post-CT [10.2% (IC 95%: 6.45-13.96) vs. 12.44% (IC 95%: 8.75-16.13) N.S.]. Comparable sperm disomic proportions were found in PRE and POST-CT samples: [(Cr.13 =0.02%, Cr.18 =0.01%, Cr.21=0.30%, Cr.XY=0.84%) vs. (Cr.13 =0%, Cr.18 =0%, Cr.21=0%, Cr.XY=0.33%), N.S]. Results were also evaluated according to the time elapsed between the PRE and POST -CT samples. No significant differences were found for seminal values (sperm concentration, p = 0.26; motility, p = 0.68), overall sperm DNA fragmentation (p = 0.68), double-strand DNA fragmentation (p = 0.90) and the frequency of sperm disomies (Cr. 13 p = 0.79, Cr.18 p = 0.46, Cr. 21 p = 0.85, Cr.XY p = 0.05). Limitations, reasons for caution The main limitations of the present study are that we only recruited patients who recovered spermatogenesis and that our data evaluated a long-term effect of CT. Wider implications of the findings All men wishing to have their own children after gonadotoxic treatment should receive adequate counseling about the side effects of the therapy. Based on our results on sperm DNA integrity and euploidy, patients who recovered spermatogenesis should not be advised to use samples that were preserved before undergoing CT. Trial registration number na

Immature oocytes obtained after ovarian stimulation can reach the metaphase II stage through in vitro maturation (IVM) and subsequently be microinjected, cleaved in vitro and give rise to a live birth. As some publications have suggested embryos derived from IVM oocytes lead to poor reproductive outcomes, immature oocytes are routinely discarded in most infertility centers. Since embryo culture conditions have improved in the last years, how to manage immature oocytes should be re-evaluated. Our study aimed to compare the outcomes of human embryos derived from IVM oocytes against those obtained by their IVO sibling oocytes. This retrospective study includes 63 cycles of patients who underwent an ICSI attempt at our center between May 2021 and December 2022. Following internal guidelines, when less than 6 oocytes were collected and/or less than 50% were mature, IVM was performed if immature oocytes were available. Overall, 489 oocytes were retrieved from 63 patients (38.7±3.3 years). After denudation, the maturity was classified as germinal vesicle (GV), metaphase I (MI), or metaphase II (MII). MII oocytes were considered in vivo mature oocytes (IVO) and were microinjected. The following day, the immature oocytes that reached the MII stage were microinjected. All embryos were cultured until day 5-6 of development. Fertilization, cleavage, blastulation and euploidy rates were compared between IVM and IVO. Initially, 171 oocytes (35.0%) were IVO, 51 (10.4%) were MI, 208 (42.5%) were GV, and the remaining 59 (12.2%) were atretic or empty zona pellucidas. After incubation, MI oocytes were more prone to reach the MII stage (n=35, 68.6%) than GV oocytes (n= 77, 37%), p<0.005. The fertilization rates for the MII coming from MI, GVs and IVO oocytes were 54.3%, 57.1% and 75.4%, respectively (p<0.05). The cleavage rates were similar in embryos coming from MI, GVs and IVO (100%, 95.4%, 99.2%, N.S.). Lower blastulation rates were observed in the embryos coming from MI and GVs vs. the rate obtained in the IVO group (15.8%, 27.3%, 59.7%, p<0.005). Overall, 14 patients (22.2%) obtained 1 additional blastocyst from IVM oocytes in their cohort, and in 3 of them (21.4%) the only usable blastocyst was generated by IVM. PGT was performed in 39 cycles due to Advanced Maternal Age. In these cycles, 2 blastocysts coming from MI were biopsied and both were chromosomally abnormal (100%). 8 blastocysts derived from GVs were biopsied, and 4 of them (50%) were aneuploid. A total of 10 blastocysts coming from IVO oocytes were analyzed, and 3 were chromosomally abnormal (30.0%). IVM oocytes lead to lower fertilization and blastulation rates, although they have similar cleavage and aneuploidy rates as their sibling embryos coming from IVO oocytes. Since there is a likelihood of obtaining euploid blastocysts from IVM oocytes, immature oocytes should not be discarded when the number of available oocytes is small.

Abstract Study question Is there evidence of cell cycle checkpoint activation in preimplantation embryos with double stranded breaks (DSBs) in their DNA, induced using CRISPR? Summary answer Cells with persistent double strand DNA damage exhibit longer timeframes to reach key morphological milestones than those with successful repair, consistent with checkpoint activation. What is known already In most cell types, DNA damage leads to activation of cell cycle checkpoints that halt the cycle while DNA repair is attempted. It has been hypothesised that early embryos lack (or have relaxed) checkpoints, and that this may be necessary to allow the embryo to divide rapidly and synchronously during the first few mitoses. It is thought that maternal (oocyte derived) inhibitors may be responsible for suppressing checkpoints until activation of the embryonic genome. Inadequate checkpoint control may be responsible for the genetic instability and sensitivity to DNA damage seen during early preimplantation stages, which is of clinical importance. Study design, size, duration 84 embryos were generated for research in an IRB approved study. For this purpose, donor oocytes were fertilised with donor sperm using ICSI. 51 of the resulting embryos served as controls, while in the other 33 double strand DNA breaks were created in a highly controlled fashion, directed at specific genomic sites using CRISPR-Cas9 technology. Successfully fertilised oocytes underwent culture in a time-lapse incubator and the duration of key developmental events were carefully timed. Participants/materials, setting, methods Precise induction of DNA damage involved injection of CRISPR-Cas9 ribonuclear complex (RNP), along with the sperm at the time of fertilization (ICSI). All embryos were disaggregated on day-3 and their cells subjected to whole genome amplification. Amplified products underwent low-pass next generation sequencing to detect segmental aneuploidy related to failure of DNA repair, while the site targeted using CRISPR-Cas9 was PCR amplified and sequenced to confirm whether it had successfully undergone repair. Main results and the role of chance Embryos that successfully repaired the induced DNA damage showed timings of pronuclei appearance/disappearance and cell divisions that were indistinguishable from control embryos. Rates of embryo arrest prior to completion of the second mitotic division were also similar between the two groups (25% versus 24%). In contrast, embryos with unresolved DNA damage, as evidenced by the detection of chromosomal fragments involving a breakpoint at the site targeted using CRISPR, were delayed in reaching the same developmental milestones (p < 0.0001 for time to first cleavage division) and displayed a much higher incidence of arrest (63% before the second mitotic division; p = 0.0002). The delay in mitotic progression in blastomeres with DNA damage represents strong evidence that a checkpoint is active in the cells of cleavage stage embryos, sensing double strand DNA breaks and slowing the cell cycle. However, the fact that 36% of embryos with unresolved DNA damage continued to progress, albeit at a slower rate, is consistent with the notion that checkpoint control is less stringent in early human embryos. This relaxation of control is likely to contribute to the genetic instability seen at the cleavage stage, including a risk of unresolved DNA damage and a high frequency of chromosomal malsegregation (causing mosaicism). Limitations, reasons for caution While altered cell cycle timings are consistent with checkpoint activation, other factors could conceivably influence the duration of key morphokinetic events. Complimentary experiments to test the functionality of specific checkpoints and/or the activity of their individual components will be required for definitive proof that embryonic checkpoints are active but weakened. Wider implications of the findings Segmental aneuploidy, micronucleation and mosaicism are frequently seen in human preimplantation embryos and are associated with reduced likelihood of ongoing pregnancy. Such abnormalities are symptomatic of genetic instability associated with excessively permissive checkpoints. These results highlight the need for culture systems that protect embryos from cellular stressors, especially DNA damage. Trial registration number N.A.

Recent studies have indicated that around 50% of transgender adolescents express a desire to have children in adulthood. During gender transition, information about the choices they have on fertility preservation and ART is crucial. This study aims to describe the transgender population attending our fertility clinic, the treatments of choice and their clinical outcomes. This is an observational study including 56 transgender individuals that attended our clinic between 2010-2022. Individual's sense of transition, age at treatment, sexual orientation, occurrence of a gender-affirming surgery (GAS) and/or the administration of gender-affirming hormonal therapy (GAHT) was described. The increase in the number of treatments over the years, the type of fertility treatments they underwent, and their clinical outcomes were also analysed. Fifty-four transmen and 2 transwomen were included in this study (mean age at treatment was 29.0±6.7 years). Information about sexual orientation was gathered for 44 transmen and all of them (100%) declared themselves to be heterosexual. GAS had been undergone by 22 (40.7%) transmen including bilateral mastectomy (n=10) and hysterectomy-bilateral anexectomy-bilateral mastectomy (n=12). Concerning GAHT, 26 (48.1%) were following a hormonal therapy either with testosterone (n=24) or oestrogen (n=2). The overall number of ART treatments in this community increased 5.6-fold during the period of 2018-2022 in relation to the 2010-2017 period, and a total of 93 fertility treatments were carried out. Regarding the distribution of treatments, the majority of patients underwent cycles involving their cis partner's and sperm banking [n= 64 cycles (68.8%) including intrauterine insemination (n=51, 54.8%), ICSI (n=6, 6.5%) and third party oocyte donation (n=7, 7.5%)] obtaining clinical pregnancy rates comparable to the general population attending our clinic (33.3%, 71.4%, 50.0%, respectively). Eight transmen underwent 12 (12.9%) ovarian stimulations for an oocyte donation to their cis partner with a pregnancy rate of 55%. Finally, 16 individuals carried out 17 fertility preservation procedures [14 oocyte vitrification (15.0%) and 3 sperm freezing (3.3%)]. Mean number of oocytes obtained after ovarian stimulation was 11.2± 6.9 in transmen, and the mean sperm parameters in the transwomen were as follows: 0.5±0.5 ml; 2± 2 M/ml total count; 16± 14% motility. Our results confirm a recent increase in the number of transgender individuals attending fertility clinics. This transgender population is characterised by being mostly heterosexual transmen in their thirties who have undergone hormonal and/or surgical transition. These characteristics may determine their preference for a treatment on their cis partner, preferably intrauterine insemination using sperm bank.

Abstract Study question Is double-strand DNA break repair functional in human preimplantation embryos and what implications might a deficiency have for genome editing performed at early developmental stages? Summary answer CRISPR-Cas9 succeeds in generating DNA breaks with high efficiency, but these often remain unresolved, indicating that early human embryos are deficient in DNA repair. What is known already Genome editing (GE) technologies, such as CRISPR-Cas9, raise the possibility of avoiding transmission of inherited disorders by converting mutant genes back to wild-type (normal). The therapeutic application of CRISPR-Cas9 at the preimplantation stage is controversial due to uncertainty over safety and concerns about the alteration of the human germline. However, from a technical perspective it remains highly attractive, since it is the only developmental stage where delivery of CRISPR-Cas9 to every cell of the individual can be guaranteed. Currently, information on safety aspects and efficacy are lacking. Therefore, evaluation of the cellular response to CRISPR-Cas9 in human embryos is needed. Study design, size, duration 84 embryos were generated for research in an IRB approved study. For this purpose, donor oocytes were fertilised with donor sperm using ICSI. 51 of the resulting embryos served as controls, while in the other 33 double strand DNA breaks (DSBs) were created in a highly controlled fashion, directed at specific genomic sites using CRISPR-Cas9 technology. Successfully fertilised oocytes underwent culture in a time-lapse incubator and the duration of key developmental events were carefully timed. Participants/materials, setting, methods CRISPR-Cas9 components targeted several non-coding sites in the genome. An artificial DNA fragment was provided, which cells could potentially use for homology directed repair (HDR). Embryos were grown for 60 hours, then disaggregated. Blastomeres underwent whole genome amplification, followed by next generation sequencing to detect segmental aneuploidy related to failure of DNA repair. Additionally, sites targeted by CRISPR-Cas9 were amplified and sequenced to confirm whether repair had occurred and to distinguish the specific mechanism employed. Main results and the role of chance The study successfully validated and utilised three CRISPR ‘guides’ that targeted sites on chromosomes 2, 3 and 12. Alterations at the targeted sites were detected in 24/25 embryos (96% targeting efficiency). Repair of DSBs created by CRISPR-Cas9 was considered successful when an embryo contained at least one blastomere with changes to the DNA sequence at the targeted site consistent with nonhomologous end joining (NHEJ) or HDR. In total, 53 double-stranded breaks were generated by CRISPR-Cas9 during this study. Of these, 51% (27) were repaired by NHEJ, 9% (5) repaired by HDR and 40% (21) remained unresolved, the persistent breakage eventually causing segmental aneuploidy with a breakpoint at the targeted side. Segmental abnormalities are known to be detrimental to embryonic viability and risk congenital abnormalities in offspring. This study shows that human embryos, prior to activation of the genome, have a DNA damage repair deficiency. Timings of morphokinetic milestones were altered in embryos with unresolved DNA damage, confirming that checkpoint control is active. However, continued mitotic division, despite the presence of DSBs, shows cellular mechanisms that usually preserve genetic integrity lack stringency, ultimately failing to ensure repair. The results provide a warning against the therapeutic use of CRISPR-Cas9 in human embryos. Limitations, reasons for caution While targeting the embryonic genome using CRISPR-Cas9 was remarkably efficient (DSBs introduced in 96% of embryos), the majority of cells utilised NHEJ for repair, a process allowing introduction of additional mutations rather than correcting existing ones. Only <10% of cells used the HDR repair mechanism necessary for removal of mutations. Wider implications of the findings These findings reveal that cells of early human embryos are less able to process DSBs than other cell types. This suggests that embryo culture methods should give consideration to protecting the genome from damage. The results caution against clinical application of genome editing technologies that create DSBs, such as CRISPR-Cas9. Trial registration number NA

Studies have shown that infertile men are more likely to have higher concentrations of reactive oxygen species (ROS) in their seminal plasma. Furthermore, oxidative stress could be related to the presence of infection since the production of leukocytes would increase ROS. A novel technology - the MiOXSYS system-, has been developed to measure static oxidation-reduction potential (sORP) rapidly in semen, but needs to be validated. The present study aims to prove if there is any correlation between the results provided by the MiOXSYS system, the sperm parameters and the presence of both, infection and leukocytes. Prospective observational study including 52 infertile males (35years; CI95%: 34-36years) attending our clinic between 2021-2022. Inclusion criteria were: ≥1year unprotected intercourse, total motile progressive sperm ≥5million, and female partner <37years. Patients with varicocele/testicular torsion, smokers >5cigarettes/day, and previous infection or antioxidant intake were excluded. Leukocyte concentration was quantified by the ENDTZ test, and the presence of the microorganisms was assessed through a semen culture. Oxidative stress was measured with the MiOXSYS system, which provided the results of the sORP. The sORP measurement was correlated with conventional sperm parameters, leukocytes and infection, and its predictive value on leukocyte concentration and seminal culture results was also determined. Mean sORP in our patients was 1.17mV/106sperm/ml (CI95%: 0.81-1.53). A high correlation was found between sORP and sperm concentration (-0.59, P<0.0001; CI95%:-0.74 to -0.37), sperm progressive motility (-0.33, P<0.0183; CI95%: -0.55 to -0.06), total sperm motile (-0.44, P<0.0010; CI 95%: -0.64 to -0.19), and teratozoospermia rate (-0.32, P<0.0202; CI95%: -0.55 to -0.05). Leukocytes were observed in 23 (44.23%) of the samples and the mean leukocyte concentration was 0.70cells/ml (CI95%: 0.46 to 0.94). Samples with leukocytes showed a similar sORP to those without [1.22 (CI 95%: 0.60 to 1.84) vs. 1.13 mV/106 sperm/ml (CI95%: 0.70 to 1.56), N.S.]. When a ROC curve was performed to assess the ability to predict the presence of white cells, the AUC was 0.54 (CI95%: 0.40-0.68). Microorganisms were observed in 14 patients (26.90%), and they were both, cocci (n=13, 92.86%) and bacilli (n=4, 28.57%). Samples with positive spermioculture had a quite similar sORP to the negative ones [0.84 (CI95%: 0.59 to 1.09) vs. 1.29 mV/106 sperm/ml (CI95%: 0.81 to 1.77), N.S.]. The AUC for the presence of microorganisms was 0.55 (CI95%: 0.41 to 0.69). Although the oxidative stress detected through the MiOXSYS system correlates with poorer sperm variables, it doesn’t help predict the presence of leukocytes or infection in semen.

The number of individuals seeking sex hormone therapy due to gender dysphoria has been increasing in the last years as has the transgender population in need of a fertility treatment. Whether gender-affirming hormone therapy (GAHT) has an impact on IVF outcomes in transmen willing to have their own biological offspring needs to be elucidated. This study aims to evaluate if GAHT with testosterone administration prior to an ovarian stimulation in transmen patients affects the number and maturity of the oocytes recovered, the embryo development and the IVF clinical outcomes. This is a retrospective observational study including 12 IVF cycles performed after an ovarian stimulation in nine transmen patients that attended our clinics from January 2019 until March 2023. At the time of stimulation, seven patients had already initiated GAHT (study group) while two had not yet initiated testosterone treatment (control group). In all cases, sperm banking was used to fertilize the mature oocytes and the embryos generated were transferred to the corresponding ciswomen partners after a hormonal endometrium preparation. The number of oocytes recovered, the percentage of mature and fertilized eggs, the percentage of usable blastocysts and the pregnancy, miscarriage and implantation rates were calculated. Patients of the study group (transmen mean age = 30.63 ± 4.27; partners mean age = 37.36 ± 5.08) received testosterone within an interval of 12 to 47 months, and they all discontinued the treatment between 4 and 11 months prior to the oocyte stimulation. Overall, they underwent nine ovarian stimulations, and the results of their IVF cycles were as shown: 14.62 ± 6.50 oocytes retrieved; 76.92% mature oocytes; 82.22% fertilised eggs; 70.27% usable blastocyst. Seventeen embryo transfers were carried out (mean number of embryos transferred = 1.12 ± 0.33), eleven resulted in a positive pregnancy (64.70%) and implantation rate was 63.15%. To date, five healthy babies have been born and four pregnancies ended in a clinical miscarriage (36.36%). Regarding the patients in the control group (transmen mean age = 31.33 ± 0.58; partners mean age = 30.67 ± 1.53), they underwent three IVF cycles whose results were as follows: 14.00 ± 8.89 oocytes retrieved; 82.5% mature oocytes; 72.72% fertilized eggs; 45.83% usable blastocysts. Three single blastocyst transfers were performed but none of them ended in a positive pregnancy. According to our results, when discontinuing it before a fertility treatment, a prior testosterone use does not affect the ovarian, embryonic or clinical outcomes. Larger series of patients need to be analysed to provide better evidence-based conclusions about the impact of GAHT on IVF treatments, especially on the clinical outcomes.

Although it is well established that reproductive potential declines as women age, information regarding the effect of advanced paternal age (APA) is still conflicting. The use of sperm from healthy young donors has been proposed as a strategy to reduce harms associated with APA. The purpose of this study was to compare the IVF outcomes after the use of sperm samples from patients considered APA (defined as ≥ 45 years) with those found when donor sperm was used.

Research question: Can 1-day old human unfertilized oocytes activate and blastulate after exposure to calcium ionophore (Ca.I) A23187?Design: Prospective randomized trial analysis of sibling oocytes. Seventy unfertilized sibling oocytes from 24 couples were randomly split into two groups. In the treatment group, 35 oocytes were cultured with 5-mu M Ca.I A23187 for 10 min, washed and cultured until day 6 of development (D+6). The remaining 35 oocytes (control group) were similarly cultured until D+6. Activation, cleavage and blastulation rates were compared between the two groups.Results: Comparable activation rates were observed in the oocytes incubated with Ca.I A23187 and in the control group (11.4% versus 17.1%; P = 0.49). The cleavage rate observed was 45.7% in both groups. None of the embryos reached blastocyst stage.Conclusions: Activation and cleavage can occur in unfertilized oocytes after the diagnosis of failure to fertilize. Unfortunately, the prevalence of activation is not affected by exposure to Ca.I A23187. Additionally, these embryos have no tangible reproductive potential as they arrest before reaching the blastocyst stage.