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Background Non-invasive oocyte quality scoring, based on cumulus gene expression analysis, in combination with morphology scoring, can increase the clinical pregnancy (CPR) and live birth rates (LBR) in Day 3 eSET (elective single embryo transfer) ICSI patients. This was first investigated in a pilot study and is now confirmed in a large patient cohort of 633 patients. It was investigated whether CPR, LBR and time-to-pregnancy could be improved by analyzing the gene expression profile of three predictive genes in the cumulus cells, compared to patients with morphology-based embryo selection only. Methods A large interventional, non-randomized, assessor-blinded cohort study with 633 ICSI patients was conducted in a tertiary fertility center. Non-PCOS patients, 22–39 years old, with good ovarian reserve, were stimulated with HP-hMG using a GnRH antagonist protocol and planned for fresh Day 3 eSET. The cumulus cells from individually denuded oocytes were ranked by a lab-developed cumulus cell test: qRT-PCR for three predictive genes (CAMK1D, EFNB2 and SASH1) and two control genes (UBC, B2M). The embryo selected for transfer was highest ranked from the pool of morphologically transferable Day 3 embryos. Patients in the control (n = 520) and experimental arm (n = 113) were compared for clinical pregnancy and live birth, using a weighted generalized linear model, and time-to-pregnancy using Kaplan-Meier curves. Results The CPR was 61% in the experimental arm (n = 113) vs 29% in the control arm (n = 520, p p p Cumulus cell tested patients n = 65) or ≥ 35 years (n = 48) had a CPR of 62 and 60% respectively (ns). For cumulus cell tested patients with 2, 3–4, or > 4 transferable embryos, the CPR was 66, 52, and 67% (ns) respectively, and thus independent of the number of transferable embryos on Day 3. Conclusions This study provides further evidence of the clinical usefulness of the non-invasive cumulus cell test over time in a larger patient cohort. Trial registration Clinicaltrials.gov, NCT03659786/NCT02962466 (Registered 6Sep2018/11Nov2016, retrospectively registered.

STUDY QUESTION Does the freeze-all strategy in high-responders increase pregnancy rates and improve safety outcomes when compared with GnRH agonist triggering followed by low-dose hCG intensified luteal support with a fresh embryo transfer? SUMMARY ANSWER Pregnancy rates after either fresh embryo transfer with intensified luteal phase support using low-dose hCG or the freeze-all strategy did not vary significantly; however, moderate-to-severe ovarian hyperstimulation syndrome (OHSS) occurred more frequently in the women who attempted a fresh embryo transfer. WHAT IS KNOWN ALREADY Two strategies following GnRH agonist triggering (the freeze-all approach and a fresh embryo transfer attempt using a low-dose of hCG for intensified luteal phase support) are safer alternatives when compared with conventional hCG triggering with similar pregnancy outcomes. However, these two strategies have never been compared head-to-head in an unrestricted predicted hyper-responder population. STUDY DESIGN, SIZE, DURATION This study included women with an excessive response to ovarian stimulation (≥18 follicles measuring ≥11 mm) undergoing IVF/ICSI in a GnRH antagonist suppressed cycle between 2014 and 2017. Our primary outcome was clinical pregnancy at 7 weeks after the first embryo transfer. Secondary outcomes included live birth and the development of moderate-to-severe OHSS. PARTICIPANTS/MATERIALS, SETTING, METHODS Following GnRH agonist triggering, women were randomized either to cryopreserve all good-quality embryos followed by a frozen embryo transfer in an subsequent artificial cycle or to perform a fresh embryo transfer with intensified luteal phase support (1500 IU hCG on the day of oocyte retrieval, plus oral estradiol 2 mg two times a day, plus 200 mg of micronized vaginal progesterone three times a day). MAIN RESULTS AND THE ROLE OF CHANCE A total of 212 patients (106 in each arm) were recruited in the study, with three patients (one in the fresh embryo transfer group and two in the freeze-all group) later withdrawing their consent to participate in the study. One patient in the freeze-all group became pregnant naturally (clinical pregnancy diagnosed 38 days after randomization) prior to the first frozen embryo transfer. The study arms did not vary significantly in terms of the number of oocytes retrieved and embryos produced/transferred. The intention to treat clinical pregnancy and live birth rates (with the latter excluding four cases lost to follow-up: one in the fresh transfer and three in the freeze-all arms, respectively) after the first embryo transfer did not vary significantly among the fresh embryo transfer and freeze-all study arms: 51/105 (48.6%) versus 57/104 (54.8%) and 41/104 (39.4%) versus 42/101 (41.6%), respectively (relative risk for clinical pregnancy 1.13, 95% CI 0.87–1.47; P = 0.41). However, moderate-to-severe OHSS occurred solely in the group that received low-dose hCG (9/105, 8.6%, 95% CI 3.2% to 13.9% vs 0/104, 95% CI 0 to 3.7, P LIMITATIONS, REASONS FOR CAUTION The sample size calculation was based on a 19% absolute difference in terms of clinical pregnancy rates, therefore smaller differences, as observed in the trial, cannot be reliably excluded as non-significant. WIDER IMPLICATIONS OF THE FINDINGS This study offers the first comparative analysis of two common strategies applied to women performing IVF/ICSI with a high risk to develop OHSS. While pregnancy rates did not vary significantly, a fresh embryo transfer with intensified luteal phase support may still not avoid the risk of moderate-to-severe OHSS and serious consideration should be made before recommending it as a routine first-line treatment. Future trials may allow us to confirm these findings. STUDY FUNDING/COMPETING INTEREST(S) The authors have no conflicts of interest to disclose. No external funding was obtained for this study. TRIAL REGISTRATION NUMBER ClinicalTrials.gov identifier NCT02148393. TRIAL REGISTRATION DATE 28 May 2014 DATE OF FIRST PATIENT’S ENROLMENT 30 May 2014

Study question Does extended culture to the blastocyst stage affect singleton birthweight after either fresh or vitrified-warmed embryo transfer? Summary answer Singleton birthweight z-scores did not vary significantly after a fresh blastocyst transfer, whereas the additional effect of vitrification remains inconclusive. What is known already Observational studies have associated extended culture with an increased risk of preterm birth and low birthweight. On the contrary, in terms of birthweight and gestational age, singletons born after vitrification have been associated with a better perinatal outcome when compared to those born following a fresh transfer. Study design, size, duration Our post-hoc cohort analysis on neonatal outcomes included 447 liveborn singletons was derived from a recent retrospective analysis on cumulative live birth rates after cleavage-stage and blastocyst transfers. These babies were born following a fresh single cleavage-stage transfer (FCT Day 3, n = 113), fresh single blastocyst transfer (FBT Day 5, n = 218), vitrified-warmed cleavage-stage transfer (VCT Day 3, n = 58) or vitrified-warmed blastocyst transfer (VBT Day 5, n = 58). Participants/materials, setting, methods Singleton birthweight was the primary outcome measure. Gestational age and gender of the newborn were accounted for by using birthweight z-scores in a multivariable linear regression analysis, adjusting for other confounders (maternal age, BMI, parity and smoking behaviour). Vanishing twins were excluded from the analysis. Main results and the role of chance A significantly lower z-score was observed after blastocyst transfer compared to cleavage-stage transfer in the vitrified-warmed Day 5 group (P = 0.013), a difference not observed in the fresh transfer groups (P = 0.32). Following multivariable regression analysis [adjusted regression coefficient (95% confidence interval)], the FCT and FBT groups showed no significant influence on the birthweight z-scores after fresh transfer [-0.19 (-0.44; 0.05)], but the transfer of vitrified blastocysts (VBT) was associated with a lower birthweight [-0.52 (-0.90; -0.15)] compared with the transfer of vitrified cleavage-stage embryos (VCT). Limitations, reasons for caution The present cohort was relatively small, especially in the vitrified-warmed subgroups. Pregnancy-associated factors possibly influencing birthweight (such as diabetes, hypertension, pre-eclampsia) were also not accounted for in the analysis. Wider implications of the findings Different ART procedures, including extended culture and vitrification, may hold potential safety issues. These results require further confirmation in future larger studies. Study funding/competing interest(s) None. Trial registration number N/A.

Objective To study the difference in live birth rate (LBR) between frozen-warmed blastocyst transfer (FET) on the 6th or the 7th day of progesterone administration in artificially prepared cycles. Design Retrospective cohort study. Setting Tertiary university-based referral hospital. Patient(s) Patients who underwent FET between December 2015 and December 2017 in a hormone replacement therapy cycle (HRT). Intervention(s) Group A included all eligible patients who underwent transfer of a vitrified-warmed blastocyst on the 6th day of progesterone administration; group B included patients who underwent blastocyst transfer on the 7th day of progesterone. The artificial HRT protocol in this study consisted of estrogen administration at a dose of 2 mg twice daily for 7 days followed by 2 mg three times daily for 6 days and micronized vaginal progesterone 200 mg three times daily from an adequately considered endometrial thickness onward. Main Outcome Measure(s) Live birth rate. Results The study included 619 patients, 346 in group A and 273 in group B. The LBRs were comparable between both groups (36.6% for group A and group B), even after adjustment for confounding factors (adjusted odds ratio 1.073, 95% confidence interval 0.740–1.556). Subgroup analysis revealed significantly higher miscarriage rates for day 6 blastocysts transferred on the 6th day of progesterone supplementation compared with transfer on the 7th day of progesterone supplementation (50.0% versus 21.4%, respectively). Additionally, there was a tendency toward a higher LBR when the 7-day progesterone supplementation protocol was used for transfer of a day 6 blastocyst (21.5% and 35.5% for group A and group B, respectively). Conclusion Warmed blastocyst transfer on the 6th compared with the 7th day of progesterone administration in an HRT cycle results in similar LBR. Subgroup analysis of day 6 blastocysts showed significantly higher miscarriage rates when FET was performed on the 6th day of progesterone administration.

STUDY QUESTION: Do cumulative live birth rates differ between single cleavage-stage Day 3 transfer and single blastocyst-stage Day 5 transfer? SUMMARY ANSWER: Cumulative live birth rates after Day 3 and 5 transfers were similar in young patients when the vitrified embryo transfers were also taken into account. WHAT IS KNOWN ALREADY: Previous evidence has shown that the probability of live birth following IVF with a fresh embryo transfer is significantly higher after blastocyst-stage Day 5 transfer. However, because the introduction of vitrification has enhanced the survival of cryopreserved embryos and improved pregnancy rates, the optimal outcome measure for this comparison should now be cumulative live birth rates, as these include the eventual contribution of vitrified-warmed embryos. STUDY DESIGN, SIZE, DURATION: Our retrospective study included first IVF/ICSI cycles performed between January 2010 and December 2013 at a tertiary care centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients were scheduled for fresh single embryo transfer, either on Day 3 (n = 377) or on Day 5 (n = 623). Both IVF and ICSI cycles were included and the sperm used were either fresh or frozen partner ejaculates, or frozen donor ejaculates. The primary outcome was cumulative live birth (after 24 weeks) rate per started cycle, including the eventual contribution of vitrification until the birth of a first child. MAIN RESULTS AND THE ROLE OF CHANCE: Live birth rates per started cycle were significantly lower after transferring the fresh single cleavage-stage embryo, compared to a blastocyst (31.3% and 37.8%, respectively, P = 0.041). Furthermore, the number of embryo transfers necessary until the first live birth was significantly lower for blastocyst-stage embryos (P < 0.001). However, the cumulative live birth rates were 52.6% for cleavage-stage and 52.5% for blastocyst-stage transfers (P = 0.989). LIMITATIONS, REASONS FOR CAUTION: The extrapolation of the results is limited by the retrospective nature of the study. Furthermore, the analysis was restricted to patients under 36 years of age undergoing their first treatment cycle. WIDER IMPLICATIONS OF THE FINDINGS: These results deserve further clinical consideration in terms of time and cost efficiency. A subsequent analysis of the neonatal outcomes is necessary to confirm the safety of treatment cycles using extended culture. STUDY FUNDING/COMPETING INTERESTS: No external funding was received and there are no conflicts of interest to declare.

Study question Does the timing of the first frozen embryo transfer (FET) after gonadotropin-releasing hormone (GnRH) agonist triggering with the elective cryopreservation of all embryos affect pregnancy outcome? Summary answer FETs performed immediately after a freeze-all cycle did not vary significantly from delayed FETs in terms of pregnancy rates. What is known already As interest in, and use of, the freeze-all strategy expands in the field of reproductive medicine, the optimal timing to perform the subsequent FET has become increasingly important. Thus far, all clinical trials evaluating the efficacy of the segmentation strategy have opted to electively defer the first FET for at least one menstrual cycle. However, this mere empirical approach may cause unnecessary distress to infertile patients eager to conceive as soon as possible. Study design, size and duration This retrospective cohort study included the first FET cycle of all women who underwent a freeze-all protocol between October 2010 and October 2015 in two reproductive medicine centres (in Belgium and Vietnam, respectively). Participants/materials, setting and methods A total of 333 FET cycles were included in the analysis. Following the freeze-all cycle, the preparation of the endometrium consisted of the sequential administration of oestradiol valerate and micronized vaginal progesterone. The start of the FET was classified as either immediate (following the GnRH agonist withdrawal bleeding) or delayed (by at least one menstrual cycle). Clinical pregnancy rate (CPR) was the main outcome of our study. Main results and the role of chance Women in the immediate FET group were slightly younger on average (30.9 ± 4.1 versus 31.8 ± 4.3, P = 0.045) on the date of oocyte retrieval. Moreover, women in the immediate FET group received a blastocyst transfer more frequently (53.4% versus 41.6%, P = 0.038) and had fewer embryos transferred on average compared to the delayed FET group (1.8 ± 0.8 versus 2.0 ± 0.8, P = 0.013). CPR/FET was marginally significantly higher in the immediate FET group in our crude analysis (52.9% after immediate FET versus 41.6% after delayed FET, P = 0.046). In order to assess if CPR/FET remained unaltered after adjusting for measured confounding, we performed mixed-effects multivariable regression analysis. Timing of the FET no longer affected significantly the CPR of the first FET in the adjusted analysis (adjusted odds ratio (aOR): 0.62, 95% CI: 0.38-1.00; predicted CPR of 52.5% for immediate FET versus 41.8% for delayed FET). Limitations, reasons for caution The results are limited by the retrospective design and the potential for unmeasured confounding. Furthermore, we only evaluated the effect of the FET timing of the first FET on CPRs in artificially supplemented cycles and, thus, the results should not be extrapolated to live birth rates or natural-cycle FETs. Wider implications of the findings This study offers a simple but potentially relevant measure to increase patient satisfaction and adherence in couples who seek to become pregnant both safely and as soon as possible. Study funding/competing interests No funding was received for this study. The authors have no conflicts of interest to declare.

STUDY QUESTION: What is the effect of vitamin D deficiency on the pregnancy rates following frozen embryo transfer (FET)?. SUMMARY ANSWER: Vitamin D deficiency does not affect pregnancy rates in FET cycles. WHAT IS KNOWN ALREADY: Although there is evidence that the potential impact of vitamin D deficiency on reproductive outcome may be mediated through a detrimental effect on oocyte or embryo quality, the rationale of our design was based on evidence derived from basic science, suggesting that vitamin D may have a key role in endometrial receptivity and implantation. Only few retrospective clinical studies have been published to date with conflicting results. STUDY DESIGN, SIZE, DURATION: This study is the first prospective observational cohort study from the Centre for Reproductive Medicine at the University Hospital of Brussels. The duration of the study was 1 year. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 280 consecutive patients, who had at least one blastocyst frozen and were planned for a FET, were enrolled in the study following detailed information and signing of a written informed consent. Serum analysis of 25-OH vitamin D was measured on the day of embryo transfer, and the impact of vitamin deficiency was investigated on reproductive outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: Among all patients, 45.3% (n = 127) had vitamin D deficiency (

STUDY QUESTION What is the influence of vitamin D deficiency on pregnancy rates among women undergoing IVF/ICSI and Day 5 (blastocyst stage) single embryo transfer (SET)? SUMMARY ANSWER Vitamin D deficiency results in significantly lower pregnancy rates in women undergoing single blastocyst transfer. WHAT IS KNOWN ALREADY Preliminary experiments have identified the presence of vitamin D receptors in the female reproductive system. However, results regarding the effect of vitamin D deficiency on clinical outcomes are conflicting. None of the previous studies adopted a SET strategy. STUDY DESIGN, SIZE, DURATION Serum vitamin D concentration was measured retrospectively in patients who underwent SET on Day 5. Overall 368 consecutive infertile women treated within a period of 15 months were included in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS All patients underwent ovarian stimulation for IVF/ICSI and Day 5 SET. Serum samples were obtained 7 days prior to embryo transfer and stored frozen at -20°C. Samples were collectively analyzed for their 25-OH vitamin D content. Vitamin D deficiency was defined as serum 25-OH vitamin D levels

OBJECTIVE: To assess the efficiency of a "freeze-all" embryo strategy after immature oocyte retrieval (OR) and in vitro maturation (IVM) in patients with polycystic ovary syndrome (PCOS). DESIGN: Retrospective case series. SETTINGS: University-based tertiary referral center. PATIENT(S): Seventy-nine consecutive PCOS patients undergoing IVM followed by vitrified-warmed embryo transfer (ET) over a 2-year period. INTERVENTION(S): All patients received 150 IU/d highly purified hMG for 3 consecutive days. There was no hCG trigger given before OR. All day-3 embryos of good morphologic quality were vitrified. Single or double ET was performed in 114 consecutive artificial cycles. The cumulative live birth rate (LBR) per patient was calculated, as well as the projected cumulative LBR. MAIN OUTCOME MEASURE(S): LBR per patient and per retrieved immature oocyte. RESULT(S): Mean age, body mass index, and antimüllerian hormone were 28.5 ± 3.5 years, 27.8 ± 7.1 kg/m(2), and 10.3 ± 5.5 ?g/L, respectively. In total, 1,526 cumulus-oocyte complexes were retrieved. IVM yielded 800 metaphase II oocytes (52.4%), and 291 day-3 embryos were cryopreserved. Of these, 224 (76.9%) embryos were warmed. One hundred seventy-one survived (76.3%), and 105 ETs were performed. LBR per ET was 16.2% and the cumulative LBR per patient was 21.8%. LBR per retrieved immature oocyte was 1.1%. The projected LBR per patient was 24.2%. CONCLUSION(S): IVM followed by a "freeze-all" embryo strategy is a novel approach for women with PCOS. Patients who undergo IVM should be advised that each immature oocyte retrieved yields a 1.1% chance to achieve a live birth. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Objective To evaluate if increasing the interval between a failed fresh embryo transfer and a subsequent frozen embryo transfer (FET) cycle has any effect on clinical pregnancy rates (CPRs). Design Retrospective cohort study. Setting University-based tertiary referral center. Patient(s) Women who underwent at least one FET after ovarian stimulation for in vitro fertilization (IVF) and a failed fresh embryo transfer attempt from January 2010 to November 2014. We divided our sample according to the "timing" of the first FET (TF-FET), defined by the interval between oocyte retrieval and the FET cycle start date. The start of the FET was classified as either immediate (≤22 days after oocyte retrieval) or delayed (>22 days after oocyte retrieval). Intervention(s) None. Main Outcome Measure(s) CPR after the first FET. Result(s) A total of 1,183 FET cycles (performed in 1,087 women) were included in our study. No significant differences were found between the immediate and delayed FET groups regarding age, number of oocytes retrieved, number of good-quality embryos produced, embryo developmental stage at FET, and number of frozen embryos transferred. Most importantly, the CPRs of the first FET did not differ significantly according to the TF-FET (32.5% after immediate FET vs. 31.7% after delayed FET), even after adjusting for potential confounding with the use of multivariable logistic regression. Conclusion(s) FETs performed immediately after fresh IVF cycles had CPRs similar to those postponed to a later time. Therefore, deferring FETs may unnecessarily prolong time to pregnancy.

Single-embryo transfer has been proposed as a way of limiting multiple births after in vitro fertilization (IVF) in women less than 36 years of age. Extending embryo culture from the 3-day cleavage stage to the 5-day blastocyst stage might make pregnancy likelier and, in addition, the morphologic criteria that guide embryo selection are less subjective at the later date. This study analyzed the outcome of IVF in 351 infertile women age 35 and younger; 176 were assigned to have transfer of a single cleavage-stage embryo, whereas 175 received a single blastocyst-stage embryo. Multifol-licular ovarian stimulation was carried out using recombinant follicle-stimulating hormone and a gonadotropin-releasing hormone antagonist. Oocyte maturation was promoted by administering human chorionic gonadotropin, and the luteal phase was supported by vaginally administered progesterone. More than 90% of women in both treatment groups were having their first or second trial of IVF or intracytoplasmic sperm injection. The groups were similar demographically and in embryologic characteristics, but significantly more embryos in the cleavage-stage group were cryopreserved. Approximately two thirds of each group had a top-quality embryo for transfer. Compared with cleavage-stage cases, the blastocyst-stage group had a significantly higher rate of ongoing pregnancy (33% vs 22%). The relative risk (RR) was 1.54 with a 95% confidence interval (CI) of 1.08 to 2.18. Deliveries also were significantly more frequent in the blastocyst-stage group (32% vs 22%); the RR was 1.48 with a 95% CI of 1.04 to 2.11. A higher rate of first-trimester pregnancy loss in the cleavage-stage group (34% vs 19%) may be partly responsible, although the difference was not statistically significant. Two of 94 deliveries (2%), both in the cleavage-stage group, resulted in monozygotic twin births. These results affirm the effectiveness of single-embryo transfer in reducing the risk of multiple births. In younger women having their first or second trial of IVF, transfer of a single blastocyst-stage embryo is significantly likelier than use of a cleavage-stage embryo to result in pregnancy and delivery.

Objective: To compare ICSI with IVF using two insemination concentrations in moderate male infertility and to compare these data with other randomized controlled trials (RCTs). Design: Prospective, randomized, controlled trial and meta-analysis. Setting: University-based tertiary referral center. Patient(s): Seventy-three couples undergoing ART. Intervention(s): In one IVF group a standard insemination concentration of 0.2 × 10 6 /mL was used, whereas in the other group a high insemination concentration (HIC) of 0.8 × 10 6 /mL was used. Each protocol was compared with ICSI on sibling oocytes. Main Outcome Measure(s): Fertilization, fertilization failure, and embryonic development. Result(s): The overall fertilization rate was significantly lower after standard IVF than after ICSI: 37.4% vs. 64.3%. Where HIC IVF was used, the overall fertilization rate was not significantly different from that after ICSI: 59.6% vs. 67.6%. Eight randomized controlled trials were selected and reviewed together with the present randomized controlled trial by meta-analysis. The risk ratio for an oocyte to become fertilized was 1.9 (95% confidence interval of 1.4 to 2.5) in favor of ICSI, and 3.1 ICSI cycles may be needed to avoid one complete fertilization failure after conventional IVF (95% CI of 1.7 to 12.4). Conclusion(s): The data from this study and the meta-analysis show that ICSI is a more efficient technique in terms of fertilization, but not in comparison with HIC IVF.

Background In IVF-ICSI cycles with single embryo transfer (SET), embryo selection for transfer is of crucial importance. The present study aimed to define which embryo parameters might be related to the implantation potential of advanced blastocysts. Methods Overall, in 203 cycles with SET, developmental characteristics of 93 implanted (group A) and 110 non-implanted (group B) advanced blastocysts of good quality were compared. The following developmental parameters were assessed in the two groups: normal fertilization, developmental stage on day 5, number of blastomeres on day 2 and on day 3, fragmentation rate on day 3, compaction on day 4 and cleavage pattern on day 2 and day 3. Results Expanded blastocysts compared to full blastocysts have higher implantation potential (56.5% vs. 29.3%, p < 0.05). In group B, a higher proportion of advanced blastocysts showed between 10% and 50% anucleated fragments on day 3 than in group A (23.6 vs 11.8, P = 0.03). Advanced blastocysts with >10–50% fragments on day 3 showed a significant lower implantation (29.7%) than those with ≤ 10%fragments (49.4%, P = 0.03). All the other parameters analysed were comparable for the two groups. Conclusion Developmental stage on day 5 and fragmentation rate on day 3 were related to the implantation potential of advanced blastocysts and should also be taken into account in the selection of the best advanced blastocyst for transfer.