Abstract Study question What are the reproductive outcomes observed of patients who cryopreserved oocytes or embryos in the context of fertility preservation in the Netherlands? Summary answer After 10 years follow-up, 25.5% of the women used their cryopreserved oocytes or embryos to attempt pregnancy and of these, 34.6% had a live birth. What is known already Fertility preservation through freezing oocytes or embryos is an established treatment for women with a risk of premature ovarian failure (caused by a benign or oncological disease) or physiological age-related fertility decline. Little is known about the success of freezing protocols, utilization rate of oocytes or embryos and live birth rates. Study design, size, duration A retrospective nationwide observational study was performed in the Netherlands. Data were collected between 2017-2019 from all women who cryopreserved oocytes or embryos more than two years ago in the context of fertility preservation in ten out of twelve IVF centers in the Netherlands. Participants/materials, setting, methods In total 1112 women were included in this study. Medical files and patient databases were used to extract data. The study protocol was approved by the medical ethics committee. Main results and the role of chance Fertility preservation cycles are performed increasingly over the years. In the first years (2004-2007) less than 10 cycles per year were performed, which increased to more than 300 cycles per year in ten years’ time (since 2016). Initially embryos were frozen in the context of fertility preservation. In the later years cryopreservation of oocytes became the standard approach. The median number of oocytes cryopreserved was 13 per woman (range 1-52) and of embryos cryopreserved was 6 (range 1-32). Cryopreservation of oocytes versus embryos resulted in a comparable median number of eligible embryos for transfer (3 and 4 embryos respectively). The 5-year utilization rate was 12.3% and 10-year utilization rate was 25.5%. The cumulative clinical pregnancy rate after embryo transfer of cryopreserved oocytes or embryos was 39.2% and the live birth rate was 34.6% per patient. The return rate of those who had fertility preservation due to benign diseases was higher than for the other indications after five years, namely 17.1% (p < 0.05). After ten years the return rates for the other indications increased to similar percentages for all indications. Limitations, reasons for caution The follow-up period varied between patients and not all cryopreserved material was used yet at the end of this study. This might have led to underestimated pregnancy rates. Further, intention-to-treat analysis could not be performed since women who had a fertility preservation procedure without freezing were not included. Wider implications of the findings This study provides data on the reproductive outcomes after fertility preservation. This knowledge can be informative for professionals and future patients to improve counseling and informed decision making regarding ovarian stimulation in the context of fertility preservation. Trial registration number n/a

Abstract Study question Does blastocyst-stage embryo transfer in fresh and frozen cycles improve the cumulative live birth rate (cLBR) compared with cleavage-stage embryo transfer in IVF/ICSI treatments? Summary answer In good prognosis IVF patients (≥4 available embryos), a blastocyst-stage transfer policy did not result in a significant higher cLBR compared to cleavage-stage transfer policy. What is known already A recent Cochrane systematic review and meta-analysis concluded that fresh blastocyst-stage transfer in IVF/ICSI treatments is associated with higher rates of pregnancy in comparison to fresh transfer of cleavage-stage embryos. However, it is unknown whether a blastocyst-transfer policy also improves the cumulative live birth rate, i.e. the live birth rate derived from fresh and frozen-thawed embryo transfers following a single oocyte retrieval, in comparison to a cleavage-stage transfer policy in IVF/ICSI. Study design, size, duration In this multicenter randomized controlled trial women were randomly allocated to blastocyst-stage transfers (blastocyst group - fresh embryo transfer on day 5 after oocyte retrieval followed by vitrification of remnant blastocysts on day 5 and 6 following local criteria) or cleavage-stage transfers (cleavage-stage group - fresh embryo transfer on day 3 after oocyte retrieval followed by embryo cryopreservation on day 3 or 4). Randomization was stratified for age (≥36 or < 36 years). Participants/materials, setting, methods Women with a good prognosis after IVF/ICSI (defined as presenting ≥4 available embryos on day 2 of embryo culture), during their first, second, or third treatment cycle, were included. The primary outcome was the cLBR per oocyte retrieval, including associated frozen-thawed embryo transfers within 12 months after randomization (or 17 months during the COVID pandemic). Risk ratios (RR) with 95% CI adjusted for age group were calculated using log-linear binominal regression. Main results and the role of chance A total of 1202 women from 21 Dutch centers were randomly assigned to blastocyst-stage transfers (N = 599) or cleavage stage transfers (N = 603) between 2018 and 2021. At submission of this abstract, data on the primary outcome was available for 1153 (95.9%) women, 577 women in the blastocyst-stage group and 576 women in the cleavage-stage group. The cumulative live birth rate was 58.2% (336/577 women) in the blastocyst-stage group and 57.3% (330/576 women) in the cleavage stage group (RR 1.022, 95% CI 0.844-1.237; p = 0.825). The live birth rate after fresh embryo transfer was 38.0% (219/577 women) versus 29.9% (172/576 women) in the blastocyst-stage group and cleavage-stage group respectively (RR 1.282, 95% CI 1.017-1.615 p = 0.035). Interaction was found between age and day of transfer with a higher cumulative live birth rate and a higher live birth rate after fresh transfer in women of 36 years or older in the blastocyst group. Analyses on other IVF treatment outcomes, obstetrical or neonatal outcomes, patient burden, and cost effectiveness are ongoing. Limitations, reasons for caution Outcomes are only applicable for treatments of women with at least four embryos available on day two of embryo culture. Wider implications of the findings A blastocyst-stage embryo transfer policy did not result in a significant higher cumulative live birth rate in comparison to a cleavage-stage embryo transfer policy in IVF/ICSI treatments. Further research into the interaction of age with outcomes is warranted. Trial registration number NTR7034

Delivery occurred mostly by caesarean section (76.2%), indicated for PE (47.9 %), fetopelvic disproportion (50%) and for aortic rupture (one case).Two patients died from aortic rupture after caesarean section at 39 wa: the first, although concomitant emergency cardiac surgery, the other, 8 days after delivery.Both had aortic root enlargement revealed during pregnancy.61 patients gave birth to 67 newborns: one fetal demise was linked to eclampsia.Data were unavailable for 11 newborns and there are still 3 currently ongoing pregnancies.Mean weight was 2835 ± 660 gr for singletons and 2275 ± 708 gr for twins.Complications were frequent with prematurity in 25.9% and small for gestational age (< 10 th centile) in 31%.We report also a bronchopulmonary dysplasis after severe hyaline membrane disease and an acute stroke.conclusions: Pregnancies in TS after OD are at very high risk since only 33% were in our cohort associated with an absolutely normal maternofetal outcome.The most frequent maternal risk was represented by PAHD (38.5%) unrelated in our cohort with age, BMI or hGH treatment but likely linked to Turner arteropathy, small uterus and OD itself.PAHD is associated with fetal hypotrophy which represents the most frequent neonatal complication.The stress of pregnancy, especially in case of PAHD, increases the risk of aortic rupture that is already high in young women with TS as illustrated by the 2 fatal cases out of our 76 pregnancies.Our data clearly illustrate the need for tight recommendations including: 1) a systematic pregravidic cardiovascular evaluation with at least echocardiogram and/or thoracic RMN with aortic measurement normalized to body surface area; 2) complete information to patients; 3) elective single embryo transfer; 4) pergravidic follow-up in a referent centre.

BACKGROUNDThere is concern that IVF and/or ICSI might have an adverse effect on embryonic development via epigenetic alterations. Such alterations might also be involved in the sex-related growth differences in preimplantation embryos found in some animal species. In the present study we analysed cell numbers of human male and female surplus embryos that developed to the blastocyst stage after either IVF or ICSI in order to investigate possible sex-dependent differential growth rates.METHODSBlastocysts resulting from surplus embryos obtained after either IVF or ICSI during a 5 year study period were analysed using fluorescence in situ hybridization (FISH).RESULTSThe number of cells and sex could be determined in 330 blastocysts collected from 92 IVF cycles and in 322 blastocysts collected from 121 ICSI cycles. Whereas female and male embryos originating from IVF showed comparable mean log cell numbers per embryo +/- SEM (3.76+/-0.05 in 147 female and 3.72+/-0.04 in 183 male embryos), significant differences were observed in embryos originating from ICSI (3.57+/-0.05 in 162 female and 3.90+/-0.03 in 160 male embryos). The sex-related growth difference was significantly greater in ICSI than in IVF embryos. In a subset of 84 embryos, inner cell mass (ICM) and trophectoderm (TE) were analysed separately. A significantly higher mean log cell number of TE cells in ICSI male embryos was found as compared to their female counterparts (3.44+/-0.12 in 16 female and 3.90+/-0.11 in 29 male embryos), whereas this difference was not found in IVF embryos.CONCLUSIONA clear sex-related growth difference was found in human blastocysts originating from ICSI, but not in blastocysts originating from IVF. It is as yet unknown which mechanism is responsible for our findings. We hypothesize that the ICSI procedure might interfere with the process of imprinted X-inactivation.

We report on a patient with metastatic lung adenocarcinoma who underwent testing for EGFR mutations in a pleural effusion that failed to show any alterations and received standard first and second-line chemotherapy. She received erlotinib as third-line therapy with a prolonged partial response. At time of progression, re-biopsy showed squamous cell carcinoma, supported by histology and immunohistochemistry. Molecular profiling confirmed EGFR exon 21 L858R and exon 20 T790M mutations. Squamous cell transformation should not defer EGFR re-sequencing.

BACKGROUND Elective single embryo transfer (eSET) in a selected group of patients (i.e. young patients with at least one good quality embryo) reduces the number of multiple pregnancies in an IVF programme. However, the reduced overall multiple pregnancy rate (PR) is still unacceptably high. Therefore, a randomized controlled trial (RCT) was conducted comparing eSET and double embryo transfer (DET) in an unselected group of patients (i.e. irrespective of the woman's age or embryo quality). METHODS Consenting unselected patients were randomized between eSET (RCT-eSET) (n = 154) or DET (RCT-DET) (n = 154). Randomization was performed just prior to the first embryo transfer, provided that at least two 2PN zygotes were available. Non-participants received our standard transfer policy [SP-eSET in a selected group of patients (n = 100), otherwise SP-DET (n = 122)]. RESULTS The ongoing PR after RCT-eSET was significantly lower as compared with RCT-DET (21.4 versus 40.3%) and the twin PR was reduced from 21.0% after RCT-DET to 0% after RCT-eSET. The ongoing PRs after SP-eSET and SP-DET did not differ significantly (33.0 versus 30.3%), with an overall twin PR of 12.9%. CONCLUSION To avoid twin pregnancies resulting from an IVF treatment, eSET should be applied in all patients. The consequence would be a halving of the ongoing PR as compared with applying a DET policy in all patients. The transfer of one embryo in a selected group of good prognosis patients leads to a less drastic reduction in PR but maintains a twin PR of 12.9%.

BACKGROUND:Cleavage stage embryos as well as postimplantation embryos have been studied extensively over the years. However, our knowledge with respect to the chromosomal constitution of human embryos at the blastocyst stage is still rudimentary.METHODS:In the present paper, a large series of human blastocysts was examined by means of fluorescent in situ hybridization (FISH).RESULTS:It was found that only one in four blastocysts (25%) displayed a normal chromosomal pattern. We defined a group of blastocysts (26%) displaying a simple mosaic chromosome pattern (different cell lines resulting from one chromosomal error), an about equally large group of blastocysts (31%) displaying a complex mosaic chromosome pattern, and a smaller group of blastocysts (11%) showing a chaotic chromosome distribution pattern. Six per cent of all blastocysts analysed could not be assigned one of the previously mentioned chromosomal patterns.CONCLUSION:Anaphase lagging appeared to be the major mechanism through which human embryos acquire a mosaic chromosome pattern during preimplantation development to the blastocyst stage.

BACKGROUNDElective single embryo transfer (eSET), applied in the first or second IVF cycle in young patients with good quality embryos, has been demonstrated to lower the twin pregnancy rate, while the overall pregnancy rate is not compromised. It is as yet unclear whether eSET could be the preferred transfer policy in all treatment cycles, or that it should be restricted to the first or first two cycles.METHODSeSET policy (when two or more embryos were available, at least one of them being of good quality) was offered to patients younger than 38 years in the first three treatment cycles. Retrospectively, treatment cycle outcome was studied.RESULTSIn 326 patients, 586 treatment cycles were performed (326 first, 168 second and 92 third treatment cycles). In 65 cycles (11%), eSET could not be applied because there was either no fertilization, or only one embryo available. In the remaining 521 cycles, eSET was performed in 111 cycles (19%), while in 410 cycles, no good quality embryo was available resulting in the transfer of two embryos (double embryo transfer, DET). No significant differences in ongoing pregnancy rates after transfer of fresh embryos were observed between eSET and DET in the first (both 33%), second (36 and 23%, respectively) and third treatment cycles (20 and 24%, respectively). In significantly more eSET cycles compared to DET cycles, could embryos be frozen. This resulted in a significantly higher cumulative pregnancy rate after eSET compared to DET.CONCLUSIONSIn patients younger than 38 years with at least one top quality embryo, eSET can be the transfer policy of choice in at least the first three treatment cycles, since the pregnancy rates obtained in each treatment cycle are comparable to those after DET.

BACKGROUND Although well defined for embryos at cleavage stages, the occurrence and frequency of chromosomal aberrations in human blastocysts is relatively unknown. It has been reported that only one in four blastocysts is comprised totally of chromosomally normal cells. One of the selection mechanisms for the embryo proper to become free of these chromosomally abnormal cells would be to sequester them to the extra-embryonic compartment during development. The study aim was to investigate whether such a mechanism of selection exists in human preimplantation embryos. METHODS Inner cell mass (ICM)/trophectoderm (TE) differentiation was performed, followed by fluorescence in-situ hybridization (FISH), to study the chromosomal distribution in both populations of cells. RESULTS Of the 94 successfully analysed blastocysts, 68.8 +/- 1.5% of all analysable nuclei per blastocyst showed a disomic chromosomal content. Only 22.6% of blastocysts analysed were classified as normal. Of the embryos classified as abnormal at the blastocyst stage, 11.9% showed a simple mosaic pattern and 32.1% a complex mosaic pattern. An equally large group of blastocysts showed either a chaotic pattern (16.7%), or the chromosomal pattern could not be classified. The average degree of normal cells in the ICM (67.9%) was similar to the degree observed in the TE (69.5%). CONCLUSIONS These findings indicate that chromosomally abnormal cells are not preferentially segregating to the extra-embryonic compartment of the human preimplantation embryo at the blastocyst stage. Hence, other mechanisms should be responsible for an absence of chromosomally abnormal cells in the embryo proper at later stages of development. One possible mechanism might be the elimination of the chromosomally abnormal cells by selective cell death activation.