Perspectives on sperm donor anonymity: insights from donor-conceived adults in Belgium.

STUDY QUESTION: Are donor-conceived adults in Belgium interested in obtaining donor information, and do these interests vary based on their family backgrounds? SUMMARY ANSWER: Donor-conceived adults express a significant interest in obtaining donor-related information, with the highest interest reported by offspring from heterosexual couples compared to those from lesbian couple-parented or single-parent families. WHAT IS KNOWN ALREADY: In Belgium, sperm donation is mainly anonymous, but the rise of direct-to-consumer genetic testing challenges this anonymity. STUDY DESIGN, SIZE, DURATION: This was a cross-sectional study involving an online nationwide survey conducted from July 2022 to October 2023. Participants, aged 18 years and older and being aware of their anonymous sperm donor-conceived status, were recruited through various channels. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 203 participants were included: 62.6% grew up in heterosexual families with infertile fathers, 26.1% with lesbian couples, 8.4% with single parents, and 3.0% in various or diverse family structures. The survey was available in both French and Dutch and consisted of 43 questions, including a mix of yes/no questions and multiple-choice items. MAIN RESULTS AND THE ROLE OF CHANCE: The average age of disclosure was 16.5 years, with notably later disclosure in heterosexual couple-parented households. A substantial 82.8% of donor-conceived individuals expressed a keen interest in obtaining non-personally identifiable donor information, while 69% were curious about personally identifiable donor data. Furthermore, 61.6% conveyed a desire for personal contact with their donors, and 26.6% advocated for the inclusion of the donor's name on their birth certificates. Participants raised in lesbian two-parent families exhibited the lowest level of interest in donor-related information compared with those raised in other family structures. An overwhelming 90.1% wondered about the possibility of having half-siblings from the same sperm donor. Analysis of survey responses on DNA database registration revealed that 55.2% of donor-conceived offspring were already registered, with 68.8% discovering the same donor offspring and 30.4% successfully locating their donors. Compared to individuals from other family structures, those raised in heterosexual couple-parented households exhibit a less positive attitude toward their conception through anonymous sperm donation. About 61.6% of donor-conceived individuals reported experiencing distinct emotions compared to their peers, while 44.1% encountered psychological difficulties related to anonymous sperm donation, primarily attributed to late disclosure. The majority supported the idea of informing the donor about the number of children he facilitated to conceive. Lastly, the study highlighted that 21.2% of donor-conceived adults considered becoming donors themselves, and 31.3% expressed willingness to use an anonymous donor whenever faced with fertility challenges. LIMITATIONS, REASONS FOR CAUTION: Our sample size may not fully represent all adults conceived through anonymous sperm donation in Belgium. Participation bias may have influenced the results, especially due to the overrepresentation of participants from heterosexual couples. Additionally, an association exists between individuals raised by heterosexual couples and late disclosure, complicating the analysis by introducing a confounding factor. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study contribute to a better understanding of the needs and preferences of donor-conceived adults, with significant potential impact on patient education and healthcare policy. STUDY FUNDING/COMPETING INTEREST(S): Study funding was not obtained for this research. There are no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Oocyte donors' experience and expectations in a non-profit fertility care setting.

PURPOSE: In this survey, we aimed to provide the description of previous oocyte donors' profile in a Belgian tertiary fertility hospital clinic. The research question is as follows: could certain aspects be changed or improved, according to previous oocyte donors? The final purpose is to boost adherence to future oocyte donation (OD) programs, given the large gap between supply and demand. METHODS: We set up an observational cross-sectional study of oocyte donors who were recruited in a tertiary referral hospital. Participants were asked to join an anonymous online survey with questions about demographic and reproductive variables, reasons to start or discontinue OD, satisfaction rate, experience, and attitude towards presumed anonymity. RESULTS: A total of 218 women were eligible to join the study, with a response rate of 49% (108/218). The emerging profile of the oocyte donor is a well-educated (102/108 with at least a high school degree), employed (86/108) woman in her thirties. Altruism and solidarity were the main drivers of their choice (105/108), and a general permissive attitude towards disclosure of their personal information to the recipient (60/108) was registered. In case of negative experience or discontinuation, concerns regarding pain management and specific long-lasting psychological support were expressed (8/20). CONCLUSIONS: Our findings suggest the need to improve pain relief and to offer psychological support even beyond ending the donation process. These interventions could improve both participation and adherence to OD programs, ensuring an autonomous and free choice while avoiding any risk of exploitation.

Optimized sperm selection: a highly efficient device for the isolation of progressive motile sperm with low DNA fragmentation index.

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.

Early versus late follicular phase ovarian stimulation: a randomized controlled trial.

RESEARCH QUESTION: Is late follicular phase stimulation as efficient as early follicular phase stimulation in a gonadotrophin-releasing hormone (GnRH) antagonist protocol in oocyte donors in terms of the number of oocytes. DESIGN: In this open label, phase 3, non-inferiority, randomized controlled trial using a two-arm design with a 1:1 allocation ratio, 84 oocyte donors were allocated to the early follicular start group (control group, n = 41) or the late follicular start group (study group, n = 43). In the control group, women followed a fixed GnRH antagonist protocol with recombinant FSH (r-FSH) 225 IU. In the study group, r-FSH 225 IU was initiated in the late follicular phase. The primary outcome was the number of oocytes. The secondary outcomes were the number of mature oocytes, consumption of gonadotrophins and GnRH antagonist, and cost of medication. RESULTS: The number of oocytes did not differ between the control group and the study group (intent-to-treat analysis 15.5 ± 11.0 versus 14.0 ± 10.7, P = 0.52; per-protocol analysis 18.2 ± 9.7 versus 18.8 ± 7.8, P = 0.62). In addition, the number of mature oocytes did not differ between the groups (14.1 ± 8.1 versus 12.7 ± 8.5, P = 0.48). The duration of stimulation was shorter in the control group (10.0 ± 1.4 versus 10.9 ± 1.5 days, P = 0.01). The total amount of r-FSH used was lower in the control group (2240.7 ± 313.9 IU versus 2453.9 ± 330.1 IU, P = 0.008). A GnRH antagonist was used for approximately 6 days in the control group, while a GnRH antagonist was only prescribed for one woman in the study group (6.0 ± 1.4 days versus 0.13±0.7 days, P < 0.001). There was a significant difference in the cost of medication per cycle between the groups (1147.9 ± 182.8€ in control group versus 979.9 ± 129.0€ in study group, P < 0.001). CONCLUSIONS: Late follicular phase stimulation is as efficient as early follicular phase stimulation in terms of the number of oocytes.

Overview of the current situation of embryo donation in Belgian fertility centers.

OBJECTIVES: The aim of this study is to survey the current situation in Belgium regarding embryo donation (ED) practices and to explore the potential reasons for not offering this treatment option. STUDY DESIGN: A questionnaire was sent to all fertility centers in Belgium that are allowed to perform IVF regarding whether or not they perform ED for third parties, their overall experience with ED and the possible reasons for not doing it. The questionnaire was divided into three different sections, depending on whether the center currently performs ED for third parties, has never performed it or once performed it but no longer does. All respondents were anonymized. RESULTS: The questionnaire was returned by 16 out of 18 centers. Only three out of 16 centers currently perform ED. All these centers require additional actions before ED can be performed. Sometimes ED is not performed although it was indicated in the contract; the most important reasons are the need for additional investigations, the administrative obstacles and the non-eligibility of the embryos. Between 2017 and 2021, few ED were performed in these centers (n = 2, 38 and 6). Eight out of 16 centers previously offered ED but ceased. In two centers, patients who want to donate their supernumerary embryos are referred for treatment to a center where ED is performed, but none of these centers transfer embryos to a center performing ED. The main reasons for discontinuing ED were the additional investigations required and the unprofitable investment in time and personnel. Five out of 16 centers never offered ED. At one center, patients who still indicate ED for their supernumerary embryos are referred to a center performing ED. The reduction of the administrative burden and avoiding additional testing are the most indicated measures that could facilitate the introduction of an ED program. CONCLUSIONS: Embryo donation, although legally allowed, is currently hardly performed in Belgium. The reasons for this are mainly associated to additional mandatory post-hoc testing and the extra administrative burden which is not financially covered. Poor transparency and communication between Belgian centers may be an additional factor explaining the country's low embryo donation rate.

Children born after assisted reproduction more commonly carry a mitochondrial genotype associating with low birthweight.

Children conceived through assisted reproductive technologies (ART) have an elevated risk of lower birthweight, yet the underlying cause remains unclear. Our study explores mitochondrial DNA (mtDNA) variants as contributors to birthweight differences by impacting mitochondrial function during prenatal development. We deep-sequenced the mtDNA of 451 ART and spontaneously conceived (SC) individuals, 157 mother-child pairs and 113 individual oocytes from either natural menstrual cycles or after ovarian stimulation (OS) and find that ART individuals carried a different mtDNA genotype than SC individuals, with more de novo non-synonymous variants. These variants, along with rRNA variants, correlate with lower birthweight percentiles, independent of conception mode. Their higher occurrence in ART individuals stems from de novo mutagenesis associated with maternal aging and OS-induced oocyte cohort size. Future research will establish the long-term health consequences of these changes and how these findings will impact the clinical practice and patient counselling in the future.

IVF with reception of oocytes from partner in lesbian couples: a systematic review and SWOT analysis.

The growing utilization of assisted reproductive technology (ART) by the LGBTQ+ community, especially among lesbian couples, challenges societal norms and promotes inclusivity. The reception of oocytes from partner (ROPA) technique enables both female partners to have a biological connection to their child. A systematic review was conducted of the literature on ROPA IVF to provide the latest data and a SWOT analysis was subsequently performed to understand the strengths, weaknesses, opportunities and threats associated with ROPA IVF. Publications from 2000 to 2023 with relevant keywords were reviewed and 16 records were included. Five studies provided clinical information on couples who used ROPA IVF. ROPA IVF provides a unique opportunity for a biological connection between the child and both female partners and addresses concerns related to oocyte donation and anonymity. Weaknesses include limited cost-effectiveness data and unresolved practical implications. Opportunities lie in involving both partners in parenthood, advancing ART success rates and mitigating risks. Threats encompass increased pregnancy complications, ethical concerns, insufficient safety data, legal or cultural barriers, and emotional stress. In conclusion, ROPA IVF offers a promising solution for lesbian couples seeking to create a family in which both partners want to establish a biological connection with their child.

Circadian serum progesterone variations on the day of frozen embryo transfer in artificially prepared cycles.

RESEARCH QUESTION: What is the intra-day variation of serum progesterone related to vaginal progesterone administration on the day of frozen embryo transfer (FET) in an artificial cycle? DESIGN: A prospective cohort study was conducted including 22 patients undergoing a single blastocyst artificial cycle (AC)-FET from August to December 2022. Endometrial preparation was achieved by administering oestradiol valerate (2 mg three times daily) and consecutively micronized vaginal progesterone (MVP; 400 mg twice daily). A blastocyst FET was performed on the 6th day of MVP administration. Serum progesterone concentrations were measured on the day of transfer at 08:00, 12:00, 16:00 and 20:00 hours. The first and last blood samples were collected just before MVP was administered. RESULTS: The mean age and body mass index of the study population were 33.95 ± 3.98 years and 23.10 ± 1.95 kg/m2. The mean P-values at 08:00, 12:00, 16:00 and 20:00 hours were 11.72 ± 4.99, 13.59 ± 6.33, 10.23 ± 3.81 and 9.28 ± 3.09 ng/ml, respectively. A significant decline, of 2.41 ng/ml (95% confidence interval 0.81-4.00), was found between the first and last progesterone measurements. CONCLUSION: A statistically significant intra-day variation of serum progesterone concentrations on the day of FET in artificially prepared cycles was observed. This highlights the importance of a standardized procedure for the timing of progesterone measurement on the day of AC-FET. Of note, the study results are applicable only to women using MVP for luteal phase support; therefore it is necessary to confirm its validity in comparison with the different existing administration routes of progesterone.

Clinical pregnancy rate for frozen embryo transfer with HRT: a randomized controlled pilot study comparing 1 week versus 2 weeks of oestradiol priming.

RESEARCH QUESTION: Does a frozen-embryo transfer in an artificially-prepared endometrium (FET-HRT) cycle yield similar clinical pregnancy rate with 7 days of oestrogen priming compared to 14 days? DESIGN: This is a single-centre, randomized, controlled, open-label pilot study. All FET-HRT cycles were performed in a tertiary centre between October 2018 and January 2021. Overall, 160 patients were randomized, with a 1:1 allocation, into two groups of 80 patients each: group A (7 days of E2 prior to P4 supplementation) and group B (14 days of E2 prior to P4 supplementation). Both groups received single blastocyst stage embryos on the 6th day of vaginal P4 administration. The primary outcome was the feasibility of such strategy assessed as clinical pregnancy rate, secondary outcomes were biochemical pregnancy rate, miscarriage rate, live birth rate and serum hormone levels on the day of FET. Chemical pregnancy was assessed by an hCG blood test 12 days after FET and clinical pregnancy was confirmed by transvaginal ultrasound at 7 weeks. RESULTS: The analysis included 160 patients who were randomly assigned to either group A or group B on the seventh day of their FET-HRT cycle if the measured endometrial thickness was above 6.5 mm. Following screening failures and of drop-outs, 144 patients were finally included both in group A (75 patients) or group B (69 patients). Demographic characteristics for both groups were comparable. The biochemical pregnancy rate was 42.5% and 48.8% for group A and group B, respectively (p 0.526). Regarding the clinical pregnancy rate at 7 weeks, no statistical difference was observed (36.3% vs 46.3% for group A and group B, respectively, p = 0.261). The secondary outcomes of the study (biochemical pregnancy, miscarriage, and live birth rate) were comparable between the two groups for IIT analysis, as well as the P4 values on the day of FET. CONCLUSIONS: In a frozen embryo transfer cycle, performed with artificial preparation of the endometrium, 7 versus 14 days of oestrogen priming are comparable, in terms of clinical pregnancy rate; the advantages of a seven-day protocol include the shorter time to pregnancy, reduced exposure to oestrogens, and more flexibility of scheduling and programming, and less probability to recruit a follicle and have a spontaneous LH surge. It is important to keep in mind that this study was designed as a pilot trial with a limited study population as such it was underpowered to determine the superiority of an intervention over another; larger-scale RCTs are warranted to confirm our preliminary results. TRIAL REGISTRATION: Clinical trial number: NCT03930706.

Lineage segregation in human pre-implantation embryos is specified by YAP1 and TEAD1.

STUDY QUESTION: Which processes and transcription factors specify the first and second lineage segregation events during human preimplantation development? SUMMARY ANSWER: Differentiation into trophectoderm (TE) cells can be initiated independently of polarity; moreover, TEAD1 and YAP1 co-localize in (precursor) TE and primitive endoderm (PrE) cells, suggesting a role in both the first and the second lineage segregation events. WHAT IS KNOWN ALREADY: We know that polarity, YAP1/GATA3 signalling and phospholipase C signalling play a key role in TE initiation in compacted human embryos, however, little is known about the TEAD family of transcription factors that become activated by YAP1 and, especially, whether they play a role during epiblast (EPI) and PrE formation. In mouse embryos, polarized outer cells show nuclear TEAD4/YAP1 activity that upregulates Cdx2 and Gata3 expression while inner cells exclude YAP1 which upregulates Sox2 expression. The second lineage segregation event in mouse embryos is orchestrated by FGF4/FGFR2 signalling which could not be confirmed in human embryos; TEAD1/YAP1 signalling also plays a role during the establishment of mouse EPI cells. STUDY DESIGN, SIZE, DURATION: Based on morphology, we set up a development timeline of 188 human preimplantation embryos between Day 4 and 6 post-fertilization (dpf). The compaction process was divided into three subgroups: embryos at the start (C0), during (C1), and at the end (C2) of, compaction. Inner cells were identified as cells that were entirely separated from the perivitelline space and enclosed by cellular contacts on all sides. The blastulation process was divided into six subgroups, starting with early blastocysts with sickle-cell shaped outer cells (B0) and further on, blastocysts with a cavity (B1). Full blastocysts (B2) showed a visible ICM and outer cells referred to as TE. Further expanded blastocysts (B3) had accumulated fluid and started to expand due to TE cell proliferation and zona pellucida (ZP) thinning. The blastocysts then significantly expanded further (B4) and started to hatch out of the ZP (B5) until they were fully hatched (B6). PARTICIPANTS/MATERIALS, SETTING, METHODS: After informed consent and the expiration of the 5-year cryopreservation duration, 188 vitrified high quality eight-cell stage human embryos (3 dpf) were warmed and cultured until the required stages were reached. We also cultured 14 embryos that were created for research until the four- and eight-cell stage. The embryos were scored according to their developmental stage (C0-B6) displaying morphological key differences, rather than defining them according to their chronological age. They were fixed and immunostained for different combinations of cytoskeleton (F-actin), polarization (p-ERM), TE (GATA3), EPI (NANOG), PrE (GATA4 and SOX17), and members of the Hippo signalling pathway (YAP1, TEAD1 and TEAD4). We choose these markers based on previous observations in mouse embryos and single cell RNA-sequencing data of human embryos. After confocal imaging (LSM800, Zeiss), we analysed cell numbers within each lineage, different co-localization patterns and nuclear enrichment. MAIN RESULTS AND THE ROLE OF CHANCE: We found that in human preimplantation embryos compaction is a heterogeneous process that takes place between the eight-cell to the 16-cell stages. Inner and outer cells are established at the end of the compaction process (C2) when the embryos contain up to six inner cells. Full apical p-ERM polarity is present in all outer cells of compacted C2 embryos. Co-localization of p-ERM and F-actin increases steadily from 42.2% to 100% of the outer cells, between C2 and B1 stages, while p-ERM polarizes before F-actin (P < 0.00001). Next, we sought to determine which factors specify the first lineage segregation event. We found that 19.5% of the nuclei stain positive for YAP1 at the start of compaction (C0) which increases to 56.1% during compaction (C1). At the C2 stage, 84.6% of polarized outer cells display high levels of nuclear YAP1 while it is absent in 75% of non-polarized inner cells. In general, throughout the B0-B3 blastocyst stages, polarized outer/TE cells are mainly positive for YAP1 and non-polarized inner/ICM cells are negative for YAP1. From the C1 stage onwards, before polarity is established, the TE marker GATA3 is detectable in YAP1 positive cells (11.6%), indicating that differentiation into TE cells can be initiated independently of polarity. Co-localization of YAP1 and GATA3 increases steadily in outer/TE cells (21.8% in C2 up to 97.3% in B3). Transcription factor TEAD4 is ubiquitously present throughout preimplantation development from the compacted stage onwards (C2-B6). TEAD1 displays a distinct pattern that coincides with YAP1/GATA3 co-localization in the outer cells. Most outer/TE cells throughout the B0-B3 blastocyst stages are positive for TEAD1 and YAP1. However, TEAD1 proteins are also detected in most nuclei of the inner/ICM cells of the blastocysts from cavitation onwards, but at visibly lower levels as compared to that in TE cells. In the ICM of B3 blastocysts, we found one main population of cells with NANOG+/SOX17-/GATA4- nuclei (89.1%), but exceptionally we found NANOG+/SOX17+/GATA4+ cells (0.8%). In seven out of nine B3 blastocysts, nuclear NANOG was found in all the ICM cells, supporting the previously reported hypothesis that PrE cells arise from EPI cells. Finally, to determine which factors specify the second lineage segregation event, we co-stained for TEAD1, YAP1, and GATA4. We identified two main ICM cell populations in B4-6 blastocysts: the EPI (negative for the three markers, 46.5%) and the PrE (positive for the three markers, 28.1%) cells. We conclude that TEAD1 and YAP1 co-localise in (precursor) TE and PrE cells, indicating that TEAD1/YAP1 signalling plays a role in the first and the second lineage segregation events. LIMITATIONS, REASONS FOR CAUTION: In this descriptive study, we did not perform functional studies to investigate the role of TEAD1/YAP1 signalling during the first and second lineage segregation events. WIDER IMPLICATIONS OF THE FINDINGS: Our detailed roadmap on polarization, compaction, position and lineage segregation events during human preimplantation development paves the way for further functional studies. Understanding the gene regulatory networks and signalling pathways involved in early embryogenesis could ultimately provide insights into why embryonic development is sometimes impaired and facilitate the establishment of guidelines for good practice in the IVF lab. STUDY FUNDING/COMPETING INTERESTS: This work was financially supported by Wetenschappelijk Fonds Willy Gepts (WFWG) of the University Hospital UZ Brussel (WFWG142) and the Fonds Wetenschappelijk Onderzoek-Vlaanderen (FWO, G034514N). M.R. is doctoral fellow at the FWO. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Electronic witnessing in the medically assisted reproduction laboratory: insights and considerations after 10 years of use.

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.

Association between sequence variants in the FSHR gene and reproductive outcomes following IVF in predicted normoresponders.

RESEARCH QUESTION: Is there an association between FSHR sequence variants and reproductive outcomes following IVF in predicted normoresponders? DESIGN: Multicentre prospective cohort study conducted from November 2016 to June 2019 in Vietnam, Belgium and Spain including patients aged <38 years, and undergoing IVF with a predicted normal response with fixed-dose 150 IU rFSH in an antagonist protocol. Genotyping was performed for three FSHR (c.919A>G, c.2039A>G, c.-29G>A) and one FSHB sequence variants (c.-211G>T). Clinical pregnancy rate (CPR), live birth rate (LBR) and miscarriage rate in the first embryo transfer and cumulative live birth rate (CLBR) were compared between the different genotypes. RESULTS: A total of 351 patients underwent at least one embryo transfer. Genetic model analysis that adjusted for patient age, body mass index, ethnicity, type of embryo transfer, embryo stage and number of top-quality embryos transferred revealed a higher CPR for homozygous patients for the variant allele G of c.919A>G when compared to patients with genotype AA (60.3% versus 46.3%, adjusted odds ratio [ORadj] 1.96, 95% confidence interval [CI] 1.09-3.53). Also, c.919A>G genotypes AG and GG presented a higher CPR and LBR when compared with genotype AA (59.1% versus 46.3%, ORadj 1.80, 95% CI 1.08-3.00, and 51.3% versus 39.0%, ORadj 1.69, 95% CI 1.01-2.80, respectively). Cox regression models revealed a statistically significantly lower CLBR for c.2039A>G genotype GG in the codominant model (hazard ratio [HR] 0.66, 95% CI 0.43-0.99). CONCLUSION: These results demonstrate a previously unreported association between variant c.919A>G genotype GG and higher CPR and LBR in infertile patients and reinforce a potential role for genetic background in predicting the reproductive prognosis following IVF.

Individualized luteal phase support using additional oral dydrogesterone in artificially prepared frozen embryo transfer cycles: is it beneficial?

RESEARCH QUESTION: Does additional supplementation with oral dydrogesterone improve reproductive outcomes in patients with low serum progesterone concentrations on the day of frozen embryo transfer (FET) after artificial (HRT) endometrial preparation? DESIGN: Retrospective, single-centre cohort study including 694 unique patients performing single blastocyst transfer in an HRT cycle. For luteal phase support, intravaginal micronized vaginal progesterone (MVP, 400 mg twice daily) was administered. Serum progesterone concentrations were assessed prior to FET and outco-mes were compared among patients with normal serum progesterone (≥8.8 ng/ml) continuing the routine protocol and patients with low serum progesterone (<8.8 ng/ml) who received additional oral dydrogesterone supplementation (10 mg three times daily) from the day after FET onwards. Primary outcome was live birth rate (LBR), with a multivariate regression model correcting for relevant confounders. RESULTS: Normal serum progesterone concentrations were observed in 547/694 (78.8%) of patients who continued only MVP as planned, whereas low (<8.8 ng/ml) serum progesterone concentrations were detected in 147/694 (21.2%) patients who received additional oral dydrogesterone supplementation on top of MVP from the day after FET onwards. LBR was comparable between both groups: 37.8% for MVP-only versus 38.8% for MVP+OD (P = 0.84). The multivariate logistic regression model indicated that LBR was not significantly associated with the investigated approaches (adjusted odds ratio 1.01, 95% confidence interval 0.69-1.47, P = 0.97). CONCLUSIONS: The current findings suggest that additional oral dydrogesterone supplementation in patients with low serum progesterone concentrations at the moment of transfer could have the potential to rescue reproductive outcomes in HRT-FET cycles. This field of research, however, remains hampered by the absence of randomized controlled trials.

Micro RNA in Semen/Urine from Non-Obstructive Azoospermia Patients as Biomarkers to Predict the Presence of Testicular Spermatozoa and Spermatogonia.

About half of testicular sperm extraction (TESE) procedures in men with non-obstructive azoospermia (NOA), including men with Klinefelter syndrome (KS), are unsuccessful. To avoid unnecessary invasive surgery, biomarkers for spermatozoa were studied. In addition, markers for spermatogonia in testis tissue were explored. This study aimed to find biomarkers in the semen and/or urine of NOA patients to predict the presence of spermatogonia in the testis. Differentially expressed miRNAs were identified (1) between samples from patients with and without a positive TESE procedure as well as (2) between TESE-negative patients with and without spermatogonia. A total of thirteen upregulated miRNAs (ten in seminal plasma and three in urine) were found in the TESE-negative/spermatogonia-positive group compared to the TESE-negative/spermatogonia-negative group. These miRNAs could be potential biomarkers for spermatogonia; however, more research is necessary to validate their predictive power.

Impact of embryo vitrification on children's health, including growth up to two years of age, in comparison with results following a fresh embryo transfer.

OBJECTIVE: To assess health outcomes, including growth up to 2 years of age, in children born after embryo vitrification in comparison with children born after fresh embryo transfer. DESIGN: A prospective cohort study. SETTING: A single-center university hospital. PATIENT(S): Singletons born after the transfer of vitrified or fresh embryos, either at the cleavage or blastocyst stage between 2014 and 2018, were included. INTERVENTION(S): Multiple linear and logistic regression analyses were used to study the association between outcomes after vitrified versus fresh embryo transfer, controlling for neonatal, treatment, and maternal characteristics. Subgroup analysis according to cycle protocol (hormone replacement vs. natural cycle) and strategy (freeze-all vs. previous fresh cycle) was also performed. MAIN OUTCOME MEASURE(S): Measurements at birth and growth in infancy and childhood, as well as health outcomes, including congenital malformations, interventions, medication use, and hospitalizations are reported. RESULT(S): Birth characteristics were available for 1237 and 2063 children born after embryo vitrification and fresh embryo transfer, respectively. Follow-up data were available for 582 and 757 children at infancy and for 233 and 296 children at 2 years, respectively. Birthweight, height, and head circumference SD scores of children born after embryo vitrification were higher than children born after fresh embryo transfer, even after adjustment for neonatal, treatment, and maternal characteristics. In infancy, weight and height SD scores were larger for children born after embryo vitrification, but not after adjustment for covariates. In childhood, no differences in anthropometry were observed between the groups. Weight and height gain from birth to infancy and from infancy to early childhood were comparable between the groups. Comparable rates of severe developmental problems, hospital admissions, surgical interventions, and of chronic medication use were observed up to the age of 2 years. Subgroup analysis showed that growth parameters were not affected by the cycle protocol or strategy at any age. CONCLUSION(S): Our study indicated that embryo vitrification is associated with higher birthweight, even after controlling for confounders. However, in early childhood, anthropometry and weight and height gain was not different in children born after vitrified or fresh embryo transfer.

Transcriptomic differences between fibrotic and non-fibrotic testicular tissue reveal possible key players in Klinefelter syndrome-related testicular fibrosis.

Klinefelter syndrome (KS; 47,XXY) affects 1-2 in 1000 males. Most men with KS suffer from an early germ cell loss and testicular fibrosis from puberty onwards. Mechanisms responsible for these processes remain unknown. Previous genomics studies on testis tissue from men with KS focused on germ cell loss, while a transcriptomic analysis focused on testicular fibrosis has not yet been performed. This study aimed to identify factors involved in the fibrotic remodelling of KS testes by analysing the transcriptome of fibrotic and non-fibrotic testicular tissue. RNA sequencing was performed to compare the genes expressed in testicular samples with (KS and testis atrophy) and without (Sertoli cell-only syndrome and fertile controls) fibrosis (n = 5, each). Additionally, differentially expressed genes (DEGs) between KS and testis atrophy samples were studied to reveal KS-specific fibrotic genes. DEGs were considered significant when p < 0.01 and log2FC > 2. Next, downstream analyses (GO and KEGG) were performed. Lastly, RNA in situ hybridization was performed to validate the results. The first analysis (fibrotic vs non-fibrotic) resulted in 734 significant DEGs (167 up- and 567 down-regulated). Genes involved in the extracellular structure organization (e.g. VCAM1) were found up-regulated. KEGG analysis showed an up-regulation of genes involved in the TGF-ß pathway. The KS vs testis atrophy analysis resulted in 539 significant DEGs (59 up- and 480 down-regulated). Chronic inflammatory response genes were found up-regulated. The overlap of X-linked DEGs from the two analyses revealed three genes: matrix-remodelling associated 5 (MXRA5), doublecortin (DCX) and variable charge X-Linked 3B (VCX3B). RNA in situ hybridization showed an overexpression of VCAM1, MXRA5 and DCX within the fibrotic group compared with the non-fibrotic group. To summarize, this study revealed DEGs between fibrotic and non-fibrotic testis tissue, including VCAM1. In addition, X-linked fibrotic genes were revealed, e.g. MXRA5, DCX and VCX3B. Their potential role in KS-related testicular fibrosis needs further study.

Ultrasonographic appearance of the endometrium after saline infusion in women presenting with PRM-associated endometrial changes, due to the use of ulipristal acetate.

Ulipristal acetate (UPA), used for the treatment in women with symptomatic fibroids, is associated with endometrial changes visualised on ultrasound as thickening up to more than 16 mm in approximately 10% of the patients. Is saline infusion sonography (SIS) a good alternative for more invasive techniques, to evaluate the presence of intrauterine pathology? Ten patients, presenting with UPA associated endometrial changes at their follow up ultra-sonographic evaluation, were included. Our study demonstrated that SIS is feasible and painless in patients presenting with UPA associated endometrial changes. The thickened endometrium appears to divide at the midline, making it possible to study both layers separately and exclude any suspected intrauterine pathology. Our findings suggest that SIS may be a first choice, non-invasive, painless technique to provide a proper visualisation to rule out intrauterine pathology when UPA associated endometrial changes are diagnosed after fibroid treatment. This is especially of clinical interest in front of assisted reproductive technology treatment. Invasive techniques can be withheld for patients in whom SIS examination is not contributive.Impact StatementWhat is already known on this subject? Reversible endometrial changes after ulipristal acetate (UPA) treatment in patients with symptomatic fibroids have been described. In patients who receive UPA, especially if planned to undergo ART, assessment of potential endometrial pathology is important as such interfere with proper implantation after ART. Consequently, clinicians may consider ruling out intrauterine pathology by invasive examinations such as biopsy or hysteroscopy after visualisation of the thickened endometrium.What do the results of this study add? Saline infusion sonography (SIS) was feasible and painless in patients presenting with UPA associated endometrial changes.What are the implications of these findings for clinical practice and/or further research? SIS may be a first choice, non-invasive, painless technique to provide a proper visualisation to rule out intrauterine pathology when UPA associated endometrial changes are diagnosed after fibroid treatment. This is especially of clinical interest in front of assisted reproductive technology treatment. Invasive techniques can be withheld for patients in whom SIS examination is not contributive in excluding intrauterine pathology.

Undetectable viral RNA in follicular fluid, cumulus cells, and endometrial tissue samples in SARS-CoV-2-positive women.

OBJECTIVE: To study the presence of viral RNA in the follicular fluid, cumulus cells, and endometrial tissue samples in SARS-CoV-2-positive women undergoing assisted reproductive technology (ART). DESIGN: Prospective, single-center, observational study. SETTING: Tertiary hospital. PATIENT(S): A total of 16 patients undergoing transvaginal oocyte retrieval who had a positive SARS-CoV-2 RNA test <48 hours before the procedure. All patients underwent the retrieval between September 2020 and June 2021 and used in vitro fertilization or intracytoplasmic sperm injection. All embryos were vitrified to avoid conception during SARS-CoV-2 infection. INTERVENTION(S): Follicular fluid aspirated during oocyte retrieval, cumulus cells, and endometrial samples were analyzed for SARS-CoV-2 RNA using the RealStar SARS-CoV-2 RT-PCR-Kit1.0. MAIN OUTCOME MEASURE(S): The primary outcome parameter was the detection of viral RNA in the follicular fluid, cumulus cells, and endometrial cells. Fertilization rate, embryo developmental potential, and clinical outcome after frozen embryo transfer were secondary outcome parameters. RESULT(S): Samples from 16 patients were analyzed. Cycle threshold values of <40 were considered positive. All samples were negative for SARS-CoV-2 viral RNA. No inflammatory lesions of the endometrium were identified histologically. Fertilization rate, embryo development, and clinical outcomes after embryo transfer were reassuring. CONCLUSION(S): In women infected with SARS-CoV-2 who underwent ART, viral RNA was undetectable in the follicular fluid, cumulus cells, and endometrium. Caution is warranted in view of the small sample size, and the risk of SARS-CoV-2 affecting the embryo via ART cannot be ruled out. Adequate counseling of women and couples undergoing ART is crucial in parallel with further research on the effect of exposure of the early human embryo to SARS-CoV-2. CLINICAL TRIAL REGISTRATION NUMBER: NCT04425317.

Mitochondrial DNA variants segregate during human preimplantation development into genetically different cell lineages that are maintained postnatally.

Humans present remarkable diversity in their mitochondrial DNA (mtDNA) in terms of variants across individuals as well as across tissues and even cells within one person. We have investigated the timing of the first appearance of this variant-driven mosaicism. For this, we deep-sequenced the mtDNA of 254 oocytes from 85 donors, 158 single blastomeres of 25 day-3 embryos, 17 inner cell mass and trophectoderm samples of 7 day-5 blastocysts, 142 bulk DNA and 68 single cells of different adult tissues. We found that day-3 embryos present blastomeres that carry variants only detected in that cell, showing that mtDNA mosaicism arises very early in human development. We classified the mtDNA variants based on their recurrence or uniqueness across different samples. Recurring variants had higher heteroplasmic loads and more frequently resulted in synonymous changes or were located in non-coding regions than variants unique to one oocyte or single embryonic cell. These differences were maintained through development, suggesting that the mtDNA mosaicism arising in the embryo is maintained into adulthood. We observed a decline in potentially pathogenic variants between day 3 and day 5 of development, suggesting early selection. We propose a model in which closely clustered mitochondria carrying specific mtDNA variants in the ooplasm are asymmetrically distributed throughout the cell divisions of the preimplantation embryo, resulting in the earliest form of mtDNA mosaicism in human development.

Artificially prepared vitrified-warmed embryo transfer cycles are associated with an increased risk of pre-eclampsia.

RESEARCH QUESTION: What is the association between the development of pre-eclampsia and endometrial preparation prior to vitrified-warmed embryo transfer (frozen embryo transfer, FET)? DESIGN: A retrospective cohort study at a tertiary university-based hospital, including a total of 536 pregnant patients who underwent a FET between 2010 and 2019 and delivered in the same institution; 325 patients underwent natural cycle FET (NC-FET) and 211 artificial cycle FET (AC-FET). RESULTS: Unadjusted, the incidence of pre-eclampsia was significantly higher in AC-FET cycles than in NC-FET cycles (3.7% versus 11.8%, P < 0.001). Multivariable logistic regression analysis showed that, when adjusting for type of endometrial preparation (artificial cycle versus natural cycle), oocyte recipient cycles and African ethnicity, the risk of developing pre-eclampsia was significantly associated with artificial endometrial preparation or oocyte recipient cycles (AC-FET versus NC-FET: odds ratio 2.9, 95% confidence interval 1.4-6.0, P = 0.005). CONCLUSIONS: The current data show a higher incidence of pre-eclampsia in AC-FET versus NC-FET cycles, adding further strength to the existing data on this topic. Together, these recent findings may result in a change in clinical practice, towards a preference for NC-FET cycles over AC-FET cycles in ovulatory patients. Screening for high-risk patients and the development of strategies to mitigate their risk profile could reduce the risk of pre-eclampsia. Further understanding of the different vasoactive substances excreted by the corpus luteum is vital.