Assisted reproductive technology outcomes in women with congenital uterine anomalies: a systematic review.

PURPOSE: The present systematic review aimed to assess the fecundity of women with congenital uterine anomalies (CUAs) undergoing assisted reproductive technology (ART). METHODS: The present systematic review of the literature was reported according to the PRISMA guidelines. We systematically searched PubMed, MEDLINE, Embase and Scopus, from database inception to 17th October 2023. Studies were deemed eligible only if they included women with CUAs clearly fitting into one of the categories of the ASRM Müllerian anomalies classification 2021. RESULTS: Data relevant to the reproductive outcomes of women with CUAs who underwent ART were extracted from 55 studies. Regarding Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, studies on gestational surrogacy reported a live birth rate (LBR) ranging from 37 to 54%. Uterus transplant, although still experimental, showed promising results. Most studies reported a negative impact of unicornuate uterus and partial or complete septate uterus on both the miscarriage rate (MR) and the live birth rate (LBR). The reproductive prognosis of women with unicornuate uterus was shown to be particularly poor in case of twin pregnancy. Uterus didelphys, bicornuate and arcuate uterus seem not to negatively impact the ART reproductive outcomes. Uterus didelphys was associated with an increased risk of preterm birth (PTB), cesarean section and low birth weight (LBW). CONCLUSION: Women with CUAs should be informed regarding the impact (if any) of their congenital anomaly on both the chances of success of ART and on pregnancy-related complications. Elective single embryo transfer (eSET) should always be the first choice in patients with an increased baseline obstetric risk.

Implementing preconception expanded carrier screening in a universal health care system: A model-based cost-effectiveness analysis.

PURPOSE: The limited evidence available on the cost-effectiveness (CE) of expanded carrier screening (ECS) prevents its widespread use in most countries, including Italy. Herein, we aimed to estimate the CE of 3 ECS panels (ie, American College of Medical Genetics and Genomics [ACMG] Tier 1 screening, "Focused Screening," testing 15 severe, highly penetrant conditions, and ACMG Tier 3 screening) compared with no screening, the health care model currently adopted in Italy. METHODS: The reference population consisted of Italian couples seeking pregnancy with no increased personal/familial genetic risk. The CE model was developed from the perspective of the Italian universal health care system and was based on the following assumptions: 100% sensitivity of investigated screening strategies, 77% intervention rate of at-risk couples (ARCs), and no risk to conceive an affected child by risk-averse couples opting for medical interventions. RESULTS: The incremental CE ratios generated by comparing each genetic screening panel with no screening were: -14,875 ± 1,208 €/life years gained (LYG) for ACMG1S, -106,863 ± 2,379 €/LYG for Focused Screening, and -47,277 ± 1,430 €/LYG for ACMG3S. ACMG1S and Focused Screening were dominated by ACMG3S. The parameter uncertainty did not significantly affect the outcome of the analyses. CONCLUSION: From a universal health care system perspective, all the 3 ECS panels considered in the study would be more cost-effective than no screening.

Steps forward in embryo transfer technique: a retrospective study comparing direct versus afterload catheters at different time frames.

PURPOSE: To assess whether embryo transfer (ET) technique can influence the clinical pregnancy rate (CPR) and its correlation with the embryo transfer difficulty. DESIGN: This single center retrospective cohort analysis of fresh and frozen single blastocyst transfers performed between January 2016 and December 2021 included fresh and frozen single blastocyst transfers performed during the study timeframe. Direct technique was the only one used from January 2016 to September 2017. From September 2017 to March 2019, the choice between the two techniques was given by randomization, due to a clinical trial recruitment. From April 2019, only the afterload technique was used. Preimplantation genetic testing cycles and gamete donation procedures and cycles performed with external gametes or embryos were excluded. CPR was the primary outcome, while difficult transfer rate the secondary one. Univariate and multivariate logistic regressions were performed. RESULTS: During the period, 8,189 transfers were performed. CPR of the afterload group resulted significantly higher compared to the direct group (44.69% versus 41.65%, OR 1.13, 95% CI 1.02-1.25, p = 0.017) and the rate of difficult transfers two-thirds lower (9.06% versus 26.85%, OR 0.27, 95% CI 0.24-0.31, p < 0.001). CONCLUSION: Our study demonstrated that CPR is significantly affected by the ET technique. In particular, with the afterload protocol, both CPR and easy transfer rates increased. TRIAL REGISTRATION: http://clinicaltrials.gov registration number: NCT05364528, retrospectively registered on 3rd of May 2022.

Obstetric and perinatal outcomes following programmed compared to natural frozen-thawed embryo transfer cycles: a systematic review and meta-analysis.

STUDY QUESTION: Is there an association between the different endometrial preparation protocols for frozen embryo transfer (FET) and obstetric and perinatal outcomes? SUMMARY ANSWER: Programmed FET protocols were associated with a significantly higher risk of hypertensive disorders of pregnancy (HDP), pre-eclampsia (PE), post-partum hemorrhage (PPH) and cesarean section (CS) when compared with natural FET protocols. WHAT IS KNOWN ALREADY: An important and growing source of concern regarding the use of FET on a wide spectrum of women, is represented by its association with obstetric and perinatal complications. However, reasons behind these increased risks are still unknown and understudied. STUDY DESIGN, SIZE, DURATION: Systematic review with meta-analysis. We systematically searched PubMed, MEDLINE, Embase and Scopus, from database inception to 1 November 2021. Published randomized controlled trials, cohort and case control studies were all eligible for inclusion. The risk of bias was assessed using the Newcastle-Ottawa Quality Assessment Scale. The quality of evidence was also evaluated using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. PARTICIPANTS/MATERIALS, SETTING, METHODS: Studies were included only if investigators reported obstetric and/or perinatal outcomes for at least two of the following endometrial preparation protocols: programmed FET cycle (PC-FET) (i.e. treatment with hormone replacement therapy (HRT)); total natural FET cycle (tNC-FET); modified natural FET cycle (mNC-FET); stimulated FET cycle (SC-FET). MAIN RESULTS AND THE ROLE OF CHANCE: Pooled results showed a higher risk of HDP (12 studies, odds ratio (OR) 1.90; 95% CI 1.64-2.20; P < 0.00001; I2 = 50%) (very low quality), pregnancy-induced hypertension (5 studies, OR 1.46; 95% CI 1.03-2.07; P = 0.03; I2 = 0%) (very low quality), PE (8 studies, OR 2.11; 95% CI 1.87-2.39; P < 0.00001; I2 = 29%) (low quality), placenta previa (10 studies, OR 1.27; 95% CI 1.05-1.54; P = 0.01; I2 = 8%) (very low quality), PPH (6 studies, OR 2.53; 95% CI 2.19-2.93; P < 0.00001; I2 = 0%) (low quality), CS (12 studies, OR 1.62; 95% CI 1.53-1.71; P < 0.00001; I2 = 48%) (very low quality), preterm birth (15 studies, OR 1.19; 95% CI 1.09-1.29; P < 0.0001; I2 = 47%) (very low quality), very preterm birth (7 studies, OR 1.63; 95% CI 1.23-2.15; P = 0.0006; I2 = 21%) (very low quality), placenta accreta (2 studies, OR 6.29; 95% CI 2.75-14.40; P < 0.0001; I2 = 0%) (very low quality), preterm premature rupture of membranes (3 studies, OR 1.84; 95% CI 0.82-4.11; P = 0.14; I2 = 61%) (very low quality), post-term birth (OR 1.90; 95% CI 1.25-2.90; P = 0.003; I2 = 73%) (very low quality), macrosomia (10 studies, OR 1.18; 95% CI 1.05-1.32; P = 0.007; I2 = 45%) (very low quality) and large for gestational age (LGA) (14 studies, OR 1.08; 95% CI 1.01-1.16; P = 0.02; I2 = 50%) (very low quality), in PC-FET pregnancies when compared with NC (tNC + mNC)-FET pregnancies. However, after pooling of ORs adjusted for the possible confounding variables, the endometrial preparation by HRT maintained a significant association in all sub-analyses exclusively with HDP, PE, PPH (low quality) and CS (very low quality). LIMITATIONS, REASONS FOR CAUTION: The principal limitation concerns the heterogeneity across studies in: (i) timing and dosage of HRT; (ii) embryo stage at transfer; and (iii) inclusion of preimplantation genetic testing cycles. To address it, we undertook subgroup analyses by pooling only ORs adjusted for a specific possible confounding factor. WIDER IMPLICATIONS OF THE FINDINGS: Endometrial preparation protocols with HRT were associated with worse obstetric and perinatal outcomes. However, because of the methodological weaknesses, recommendations for clinical practice cannot be made. Well conducted prospective studies are thus warranted to establish a safe endometrial preparation strategy for FET cycles aimed at limiting superimposed risks in women with an 'a priori' high-risk profile for obstetric and perinatal complications. STUDY FUNDING/COMPETING INTEREST(S): None. REGISTRATION NUMBER: CRD42021249927.

Different actors for the same play: the impact of the embryologist performing the embryo transfer.

RESEARCH QUESTION: Does the embryologist performing the embryo transfer impact the cycle outcome, in terms of ongoing pregnancy rate (OPR)? DESIGN: This single-centre retrospective study analysed the results, corrected for main confounders, from 28 embryologists and 32 physicians who performed respectively 24,992 and 24,669 fresh embryo transfers (either at cleavage or blastocyst stage) during a 20-year period from January 2000 to December 2019, in a university-affiliated tertiary care assisted reproductive technology (ART) centre. Primary outcome was OPR, defined as the number of viable pregnancies that had completed at least 12 weeks of gestation on the total number of embryo transfers performed. The study also assessed whether the embryologist's experience, measured in terms of number of embryo transfers performed prior to the day of the procedure, had an impact on their performance. The secondary aim was to assess which variable, between the embryologist and physician, more significantly impacted OPR. RESULTS: The overall unadjusted OPR was 22.54%. The embryologist performing the embryo transfer was found to significantly affect the OPR (P < 0.0001), corrected for potential confounders. However, the physician factor made a slightly greater contribution to the model (likelihood ratio 21.86, P < 0.001 versus likelihood ratio 17.20, P < 0.0001). No significant association was found between the experience of the embryologist and OPR (P = 0.067). CONCLUSIONS: These results show how the 'human factor' influences the chances of a positive outcome in the final step of a high-tech procedure and underline the importance of implementing an operator quality performance programme (both for physicians and embryologists) to ensure the maintenance of benchmark results and eventually retrain underperforming operators.

Retrospective comparison of pregnancy outcomes of fresh and frozen-warmed single blastocyst transfer: a 5-year single-center experience.

PURPOSE: To assess whether live birth rates (LBR) and maternal/neonatal complications differed following single fresh and frozen-warmed blastocyst transfer. METHODS: The present retrospective observational study analyzed 4,613 single embryo transfers (SET) (646 fresh and 3,967 frozen) from January 1, 2014, to December 31, 2018. Fresh embryo transfer at blastocyst stage was considered according to the age of the patient and her prognosis. In case of the risk of ovarian hyperstimulation syndrome, premature progesterone rise, non-optimal endometrial growth, or supernumerary embryos, cryopreservation with subsequent frozen embryo transfer (FET) was indicated. RESULTS: No differences in LBR were recorded. Fresh embryo transfers yielded an increase both in neonatal complications OR 2.15 (95% CI 1.20-3.86, p 0.010), with a higher prevalence of singletons weighting below the 5th percentile (p 0.013) and of intrauterine growth retardation (p 0.015), as well as maternal complications, with a higher placenta previa occurrence OR 3.58 (95% CI 1.54-8.28, p 0.003), compared to FET. CONCLUSION: LBR appears not to be affected by the transfer procedure preferred. Fresh embryo transfer is associated with higher risk of neonatal complications (specifically a higher prevalence of singletons weighting below the 5th percentile and of intrauterine growth retardation) and placenta previa. Reflecting on the increased practice of ART procedures, it is imperative to understand whether a transfer procedure yields less complications than the other and if it is time to switch to a "freeze-all" procedure as standard practice. TRIAL REGISTRATION: Clinical Trial Registration Number: NCT04310761. Date of registration: March 17, 2020, retrospectively registered.

The actual impact of SARS-CoV-2/COVID-19 pandemic on IVF activity: a survey across Italian ART centers.

PURPOSE: Since the end of February 2020, SARS-CoV-2 dramatically spread in Italy. To ensure that most of National Health System (NHS) resources were employed to control the pandemic, non-urgent medical procedures (including IVF) were suspended in March 2020. Here, we aimed at assessing the impact of the restrictive measures on Italian IVF activity. METHODS: In May 2020, the Italian ART Register launched an online survey (multiple choices and open answers) across ART centers (89.0% response rate; N = 170/191) to investigate how they were facing the emergency and estimate the reduction in their activity. In February 2022, the official data of the whole 2020 were published and retrospectively analyzed. The ART cycles conducted in Italy in 2020 (67,928 by 57,423 patients) were then compared to those conducted in 2019 (82,476 by 67,633 patients). The estimates formulated through the survey were compared to the actual reduction. RESULTS: In 2020, 14,548 less IVF cycles were conducted with respect to 2019 (- 17.6% reduction). This led to 2539 fewer live births (- 19.8%) than 2019. If the reduction unveiled by the survey launched in May 2020 (i.e., - 35%) would have persisted throughout 2020, a significantly larger impact was expected (4200 less newborns). Instead, the activity was gradually recovered, and it compensated the months of greatest emergency, thus fulfilling the most optimistic scenario. CONCLUSIONS: Italy suffers from the lowest birth rate in Europe, and COVID-19 impact on IVF-derived live births testified how key ART is for Italian demographics. The government should support access to these treatments with dedicated actions.

Monozygotic twin rate among ART centers: a multicenter analysis of data from 18 Italian units.

PURPOSE: The risk of monozygotic twins (MZTs) is increased in couples undergoing assisted reproductive technology (ART) treatments. Several systematic reviews have investigated the possible determinants linked to ART, but results obtained have not been conclusive. The study aims to investigate whether the incidence of MZT differed among ART centers. METHODS: This is a multicenter retrospective cohort study using the Italian ART National Registry database and involving the centers reporting data from individual ART cycles from 2015 to 2019. To investigate the incidence of MZT, only single embryo transfer cycles were considered. Women who had sex-discordant deliveries were excluded. MZT rate was calculated as the number of multiple pregnancies (more than one gestational sac at first ultrasound) out of the total number of clinical pregnancies. A binomial distribution model was used to determine the 95% CI of the frequency of MZT. RESULTS: Eighteen centers were included, and they provided data on 10,433 pregnancies. The total number of MZT was 162, corresponding to an incidence of 1.5% (95% CI: 1.3-1.8%). The rate of MZT among centers varied between 0% (95% CI: 0.0-25.9%) and 3.2% (95% CI: 1.3-8.1%). All the 95% CIs included 1.5%, rejecting the hypothesis that the MZT rate may significantly differ among centers. CONCLUSIONS: The rate of MZT did not significantly vary among ART centers. Local factors are unlikely to explain the increased rate of MZT in ART pregnancies.

Testicular sperm extraction and intracytoplasmic sperm injection outcome in cancer survivors with no available cryopreserved sperm.

OBJECTIVE: To assess rates of successful testicular sperm retrieval and intracytoplasmic sperm injection (ICSI) outcome in cancer survivors affected by non-obstructive azoospermia (NOA) or retrograde ejaculation (RE)/failure of emission (FOE). METHODS: A retrospective analysis of cancer survivors who did not cryopreserve sperm prior to treatment undergoing testicular sperm extraction (TESE). Non-cancer NOA patients and neurologic RE/FOE were the control group. RESULTS: A total of 97 cancer survivors were offered TESE and 88 (91%) accepted. Sperm was retrieved and cryopreserved in 34/67 patients with NOA (50.7%) and in 21/21 patients affected by RE/FOE (100%). Sperm retrieval rates were similar in the control group (44.9% in NOA and 100% in RE/FOE). The ICSI cumulative pregnancy rate (60%) and live birth rate (40%) per couple in 30 NOA men did not differ from controls (50.0 and 46.5%, respectively; p = 0.399/0.670). The cumulative pregnancy rate (66.7%) and live birth rate (55.6%) in 18 RE/FOE men did not differ from the control group (38.9 and 33.3%, respectively; p = 0.181/0.315). The cancer type and the resulting infertility disorder (NOA or RE/FOE) were not associated with ICSI outcomes. Female partner age was inversely related to the cumulative live birth rate, being fourfold lower (11.5%) in women ≥ 40 years and 48.8% in younger women (p = 0.0037). CONCLUSIONS: The rate of successful TESE and the ICSI outcome in cancer survivors with NOA and RE/FOE is the same as non-cancer azoospermic patients. Female partner age (older than 40 years) was associated with a significant reduction in live birth rates after TESE-ICSI procedures.

Twenty-one year experience with intrauterine inseminations after controlled ovarian stimulation with gonadotropins: maternal age is the only prognostic factor for success.

PURPOSE: To report our experience on homologous intrauterine insemination (IUI) with gonadotropin controlled ovarian stimulation (COS) cycles and to examine different variables which could predict IUI success. MATERIALS AND METHODS: This is a retrospective analysis of IUIs performed between January 1997 and December 2017. A total of 7359 COS IUI's procedures (2901 couples) were reviewed. Clinical pregnancy, live birth rate and age, body mass index (BMI), smoking habit, duration of infertility, sperm characteristics before and after treatment (total motile count, morphology, and vitality), day 3 FSH, total gonadotropin dose, and number of follicles were assessed by multivariate logistic regression analysis, and data were expressed as odds ratio (OR). RESULTS: The mean female age at the time of COS was 35.10 ± 3.93 years. The most common single infertility diagnoses were unexplained infertility (53.55%), mild male factor (19.69%), and anovulation (10.95%). The total progressive motile sperm count (TPMC) was > 1 × 106/ml (mean 1.34 ± 1.08 × 106/ml). The clinical pregnancy rate was 9.38%, and the live birth rate was 7.19% per cycle. Twin pregnancies were 12.17%. Cumulative pregnancy was 21.89% and cumulative live birth rate was 17.58% per couple. Clinical pregnancy and live birth rates were significantly associated with female age [OR 0.97 (95% CI 0.95-0.99) and 0.95 (95% CI 0.93-0.97), respectively] and day 3 FSH [OR 0.91 (95% CI 0.87-0.94) e 0.90 (95% CI 0.87-0.94), respectively]. CONCLUSIONS: Clinical pregnancy rate and live birth rates after COS-IUIs were significantly influenced by female age and FSH levels. TRIAL REGISTRATION: Clinical trial registration number: NCT03836118.

Preimplantation Genetic Testing for Aneuploidy Improves Clinical, Gestational, and Neonatal Outcomes in Advanced Maternal Age Patients Without Compromising Cumulative Live-Birth Rate.

PURPOSE: To report the effects of blastocyst stage aneuploidy testing on clinical, gestational, and neonatal outcomes for patients of advanced maternal age undergoing IVF. METHODS: This is a single-center observational-cohort study with 2 years follow-up. The study includes a total of 2538 couples undergoing 2905 egg collections (control group), 308 (PGT-A), and 106 (drop-out group, consenting for PGT-A but withdrawing due to poor embryological outcome) RESULTS: Compared with control group, PGT-A showed improved clinical outcomes (live-birth rate per transferred embryo, LBR 40.3% vs 11.0%) and reduced multiple pregnancy rate (MPR, 0% vs 11.1%) and pregnancy loss (PL, 3.6% vs 22.6%). Drop-out group showed the worst clinical outcomes suggesting that abandoning PGT-A due to poor response to ovarian stimulation is not a favorable option. Cytogenetic analysis of product of conceptions and CVS/amniocentesis showed higher aneuploid pregnancy rates for control group regardless of embryo transfer strategy (0%, 17.9%, and 19.9%, for PGT-A, control day 5 and day 3, respectively). Multivariate analysis showed no negative impact of PGT-A-related interventions on cumulative delivery rate (26.3%, 95% CI 21.5-31.6 vs 24.0%, 95% CI 22.5-25.6 for PGT-A and control, respectively) and on neonatal outcomes. CONCLUSION: PGT-A improves clinical outcomes, particularly by reducing pregnancy loss and chromosomally abnormal pregnancy for patients of advanced maternal age, with no major impact on cumulative live-birth rate (CLBR) per egg retrieval.

Prevalence of XXY karyotypes in human blastocysts: multicentre data from 7549 trophectoderm biopsies obtained during preimplantation genetic testing cycles in IVF.

STUDY QUESTION: Which is the prevalence of a 47,XXY karyotype in human blastocysts biopsied during preimplantation genetic testing for aneuploidies (PGT-A) cycles? SUMMARY ANSWER: The prevalence of a 47,XXY karyotype amongst male blastocysts without autosomal aneuploides is ~1%. WHAT IS KNOWN ALREADY: The prevalence of Klinefelter syndrome is estimated as 0.1-0.2% in male newborns. However, the KS phenotype is extremely variable and there are men with a 47,XXY karyotype and less evident signs, who may go undetected. No risk factor for the 47,XXY karyotype in products of conception has been yet clearly defined, and no data are available regarding the prevalence of this karyotype among human preimplantation embryos. STUDY DESIGN, SIZE, DURATION: This multicentre cohort study involved 7549 blastocysts obtained during 2826 PGT-A cycles performed between April 2013 and September 2017 at six IVF clinics in Italy. PARTICIPANTS/MATERIALS, SETTING, METHODS: During 2826 PGT-A cycles, 7549 blastocysts underwent trophectoderm biopsy and quantitative-PCR-based comprehensive chromosomal testing to predict the karyotype of the corresponding embryos. The results were also presented according to ranges of maternal and paternal age at oocyte retrieval as well as sperm factor and blastocyst quality. Univariate and multivariate logistic regression analyses were conducted to investigate the correlation of possible confounding factors with the prevalence of 47,XXY karyotype. MAIN RESULTS, THE ROLE OF CHANCE: Overall, 62 blastocysts were 47,XXY or had an XXY karyotype associated with autosomal aneuploidies. After exclusion of the latter, the prevalence of a 47,XXY karyotype among male blastocysts without autosomal aneuploidies was 0.9% (n = 17/1794). A significant correlation was only found for maternal age and blastocyst quality (OR: 1.20, 95% CI: 1.01-1.42; P = 0.04 and OR: 1.6, 95% CI: 1.13-2.45; P = 0.01). LIMITATIONS, REASONS FOR CAUTION: These retrospective data have been produced based on a population of infertile couples undergoing IVF and PGT-A, and the women were mainly of advanced maternal age. Moreover, the qPCR technique is validated only to detect full-chromosome uniform aneuploidies in trophectoderm biopsies. WIDER IMPLICATIONS OF THE FINDINGS: The 0.9% prevalence of the 47,XXY karyotype among male blastocysts, when compared with the 0.1-0.2% prevalence reported in the prenatal and postnatal periods, suggests four possible scenarios that require further investigations: (i) the latter prevalence is underestimated; (ii) 47,XXY blastocysts result in a lower implantation rate than euploid embryos (estimated to be ≈50%); (iii) 47,XXY blastocysts result in a higher early miscarriage rate than euploid embryos (estimated to be ≈10%); or (iv) infertile patients of advanced maternal age and referred to IVF/PGT-A produce a higher rate of 47,XXY blastocysts. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience.

STUDY QUESTION: Can a second round of biopsy, vitrification and chromosomal testing provide a valid diagnosis where the first attempt fails? SUMMARY ANSWER: The risk of inconclusive chromosomal-assessment after trophectoderm biopsy was 2.5% but a further biopsy and vitrification-warming appeared not to impair the competence of euploid blastocysts. WHAT IS KNOWN ALREADY: The increasing implementation of multicell trophectoderm biopsy has significantly reduced the risk of inconclusive diagnosis after preimplantation-genetic-testing (PGT). Yet, few reports have defined the variables that influence the risk of failure or described the technical and clinical outcomes after re-biopsy. STUDY DESIGN, SIZE, DURATION: Retrospective multicenter study involving 8990 blastocyst biopsies conducted between April 2013 and September 2017 at six IVF centers but analyzed at a single genetic laboratory. A total of 206 blastocysts were successfully re-biopsied after warming and re-expansion, then re-vitrified. And 49 of these blastocysts were diagnosed euploid and used in single-embryo-transfers (SETs). Logistic regression analyses were conducted. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 3244 PGT-for-aneuploidies (PGT-A) cycles with a freeze-all approach, vitrification and qPCR-based analysis were performed by 2687 consenting couples. DNA amplification failure (AF) or non-concurrent data resulted in inconclusive diagnoses. In case of DNA amplification, the cellularity of the biopsy was estimated according to a previously validated method. Euploid SETs were performed. Clinical pregnancy, miscarriage, live birth rates (LBR) and perinatal outcomes were monitored. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 2.5% of trophectoderm biopsies resulted in an inconclusive diagnosis (N = 228/8990). Specifically, 2% (N = 176/8990) resulted in AF and 0.5% (N = 52/8990) in non-concurrent results. The only parameters significantly associated with inconclusive diagnoses were the IVF center and the embryo age (days) at biopsy. Among samples with successful amplification, the number of cells in the biopsy and the day of biopsy were critical to limit non-concurrent results. In total, 213 blastocysts with an inconclusive diagnosis were warmed for re-analysis and the survival rate was 96.7% (N = 206/213). The euploidy rate in blastocysts biopsied twice was 51.9% (N = 107/206) and the euploid embryos were re-vitrified. Overall, 49 euploid embryos were warmed for replacement and all survived. The LBR after SET was 38.8% (N = 19/49). No minor/major obstetrical/perinatal complication was reported. LIMITATIONS, REASONS FOR CAUTION: A single aneuploidy-testing method was adopted in this retrospective analysis. A more powered report of the clinical and obstetrical/perinatal outcomes after re-biopsied and re-vitrified blastocysts euploid SET requires a larger sample size. WIDER IMPLICATIONS OF THE FINDINGS: It is important to re-biopsy and re-vitrify undiagnosed blastocysts since healthy live births can result from them. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: None.

Associations of blastocyst features, trophectoderm biopsy and other laboratory practice with post-warming behavior and implantation.

STUDY QUESTION: Are trophectoderm biopsy or other pre-vitrification features or laboratory practices associated with differences in blastocyst post-warming behavior (degeneration, re-expansion and live birth after single embryo transfer (SET))? SUMMARY ANSWER: Blastocyst morphology, day of full development and artificial shrinkage (either laser-assisted or biopsy-induced) are the pre-vitrification parameters/practices most strongly associated with post-warming behavior and implantation potential while there was no association with trophectoderm biopsy. WHAT IS KNOWN ALREADY: Since the introduction of vitrification, the adoption of cycle segmentation, freeze-all and SET policies, as well of trophectoderm biopsy-based aneuploidy testing (i.e. pre-implantation genetic testing for aneuploidies (PGT-A)), the number of blastocysts vitrified worldwide has increased greatly. Previous studies already defined generally high blastocyst cryo-survival rates after vitrification-warming (>95%), along with a positive correlation between blastocyst re-expansion and morphology with implantation. Additionally, artificial shrinkage has been outlined as a potentially beneficial procedure, while the association between embryo cryo-survival and trophectoderm biopsy is still unclear. STUDY DESIGN, SIZE, DURATION: Cohort study conducted at two IVF centers (1 and 2). A total of 2129 consecutive SETs using vitrified-warmed blastocysts in either non-PGT or PGT-A cycles between June 2016 and August 2017 were included. A freeze-all strategy was in place and three main pre-vitrification practices were used: (i) no biopsy and no artificial shrinkage (Clinic 1); (ii) trophectoderm biopsy and vitrification of collapsed blastocyst within 30 min (Clinics 1 and 2); and (iii) no biopsy but laser-assisted artificial shrinkage (Clinic 2). The primary outcome was the blastocyst degeneration rate. Overall, 2108 surviving blastocysts were graded at 1.5 h after warming for degeneration (absent or partial) and re-expansion (full, partial or absent) grades and post-warming morphological quality. Logistic regression analyses were conducted to assess the association of any pre-vitrification feature with blastocyst post-warming behavior. The logistic regressions conducted upon live birth after either untested or euploid SET also included the post-warming characteristics. PARTICIPANTS/MATERIALS, SETTING, METHODS: Center 1 is a private IVF facility, while center 2 is the IVF facility of a University hospital. In non-PGT cycles, ICSI with blastocyst culture up to full-expansion and vitrification were performed. At center 1 the untested blastocysts were vitrified when still expanded, while at center 2 they underwent laser-assisted artificial shrinkage. In PGT-A cycles, in both clinics, trophectoderm biopsy (which involves laser-assisted shrinkage) was done without previous zona-opening on Day 3, and vitrification was performed within 30 min whilst the blastocyst remained collapsed. A qPCR-based chromosome analysis was conducted. Only SETs were performed (euploid-SET in case of PGT-A). Any cycle-, laboratory- and embryo-based feature which could impact blastocyst post-warming behavior was included in the analyses as putative confounder. MAIN RESULTS AND THE ROLE OF CHANCE: The overall degeneration rate was 1% (N = 21/2129). The results were consistent among different vitrification/warming operators or kits used, as well as any other IVF laboratory-related parameter. Blastocyst artificial shrinkage (either laser-assisted or biopsy-induced) involved a lower risk of degeneration after warming (odds ratio (OR) [95% CI] = 0.26 [0.09-0.79]). Conversely, both poor morphological quality pre-vitrification and taking 7 days to reach full blastulation resulted in a significantly higher risk (OR [95% CI] = 11.67 [3.42-39.83] and 4.43 [1.10-20.55], respectively). Importantly, trophectoderm biopsy did not show any association with blastocyst cryo-survival/degeneration. Overall 2.5% (N = 53/2108) blastocysts failed to re-expand post-warming. The only parameters significantly associated with no blastocyst re-expansion post-warming were average (OR [95% CI] = 4.96 [2.20-11.21]) or poor (OR [95% CI] = 19.54 [8.39-45.50]) morphological quality and taking 7 days to reach full blastulation (OR [95% CI] = 3.19 [1.23-8.29]), as well as prevention of spontaneous hatching pre-vitrification (OR [95% CI] = 0.10 [0.01-0.85]). The post-warming features of the survived blastocyst (i.e. degeneration and re-expansion scores and morphological quality) showed no significant association with vitrified blastocyst competence (i.e. live birth) when corrected for pre-vitrification ones (i.e. morphological quality, day of full development and, for untested SET, maternal age at oocyte retrieval). Of note, poor-quality blastocysts pre-vitrification showed a high overall cryo-survival rate post-warming 92.8% (N = 116/125), but the live birth rates were only 2.1% (N = 1/48) and 7.3% (N = 5/68) after untested and euploid SET, respectively. LIMITATIONS, REASONS FOR CAUTION: This study is not randomized and the populations of patients undergoing either non-PGT or PGT-A cycles were different. Centers 1 and 2 adopted different pre-vitrification practices for non-biopsied blastocysts, according to their own laboratory policy. To this regard, multivariate logistic regression analyses were conducted for all outcomes under investigation. WIDER IMPLICATIONS OF THE FINDINGS: Pre-vitrification features may be used to assist selection of competent embryos, moreover, these results allay concern that trophectoderm biopsy might be associated with impaired blastocyst quality or competence after vitrification/warming. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: None.

Levothyroxine supplementation on assisted reproduction technology (ART) outcomes in women with subtle hypothyroidism: a retrospective study.

The need for treating subclinical hypothyroidism (SCH) in women undergoing assisted reproduction technology (ART) is under debate. Moreover, it is known that controlled ovarian hyperstimulation (COH) protocols may impair the thyroidal axis. Therefore, we evaluated if levothyroxine (L-T4) supplementation in SCH women before undergoing ART positively affects the main reproductive outcomes. We retrospectively analyzed in vitro fertilization (IVF) data of 4147 women submitted to 6545 cycles in a tertiary care IVF Center (January 2009-December 2014). L-T4 (1.4-2.0 mcg/kg) treatment was offered to all women with a pre-cycle TSH >2.5 mIU/L before starting COH and main ART outcomes were compared in euthyroid and L-T4-treated women undergoing ART. Among 4147 women, 1074 (26%) were affected by SCH and were treated with L-T4 before COH was started. No statistically significant differences among L-T4-treated and euthyroid women group were observed regarding pregnancy rate, respectively, per cycle (27.67% vs 26.37%; p = .314) and per embryo transfer (30.13% vs 29.17%; p = .489), live birth rate, respectively, per cycle (21.58% vs 20.38%; p = .304) and per embryo transfer (23.49 vs 22.54%; p = .449) and the rest of primary and secondary efficacy endpoints. Early L-T4 treatment for infertile women with a subtle thyroid dysfunction may mitigate and protect from the negative effects of SCH in the setting of ART, and may preventively overcome also the negative impact of COH on thyroidal axis.

Correction to: Assisted reproductive technologies (ART) and childhood cancer: is the risk real?

The original version of this article unfortunately contained a mistake in the 1st sentence of the 1st paragraph in page 2.

No advantage of fresh blastocyst versus cleavage stage embryo transfer in women under the age of 39: a randomized controlled study.

PURPOSE: Is there a difference in implantation and pregnancy rates between embryos transferred electively at cleavage or blastocyst stage in infertile women ≤ 38 years with at least four zygotes on day 1 post retrieval? METHODS: A randomized clinical trial was conducted in a single tertiary care hospital with a sample size of 194 patients in each arm for a total population of 388 women. Patients less than 39 years of age with more than three fertilized oocytes and less than four previous assisted reproductive technology (ART) attempts were inclusion criteria. RESULTS: The two groups were similar for age, years of infertility, indication to treatment, basal antimüllerian hormone and FSH, number of previous ART cycles, primary or secondary infertility, type of induction protocol, days of stimulation, total gonadotrophin dose, and estradiol (E2) and progesterone (P) levels at trigger. No statistically significant differences were found in terms of number of retrieved oocytes, inseminated oocytes, fertilization rate, canceled transfers (7.73% in blastocyst and 3.61% in cleavage stage group), and cycles with frozen embryos and/or oocytes. Although a higher number of fertilized oocytes were in the blastocyst stage group (6.18 ± 1.46 vs 5.89 ± 1.54, p = 0.052), a statistically greater number of embryos/randomized cycle were transferred at cleavage stage (1.93 ± 0.371) compared with the number of transferred blastocysts (1.80 ± 0.56), probably due to the number of embryos not reaching blastocyst stage (3.09%). The implantation rate (28.37 vs 25.67%), pregnancy rate per cycle (36.06 vs38.66%), transfer (39.66 vs 40.11%), spontaneous abortions (19.72% vs 12.00%), delivery rate per cycle (27.84 vs 32.99%), and transfer (30.17 vs 34.22%) were not significantly different between the blastocyst and cleavage stage groups. The twin delivery rate was higher in the blastocyst stage group, although not significant (42.59 vs 28.12%). The mean numbers of frozen blastocyst (2.30 ± 1.40 vs 2.02 ± 1.00) and frozen oocytes (7.09 ± 3.55vs 6.79 ± 3.26) were not significantly different between the two groups. CONCLUSIONS: Fresh blastocyst-stage transfer versus cleavage-stage transfer did not show any significant difference in terms of implantation and pregnancy rate in this selected group of patients. A high twin delivery rate in both groups (35.59%) was registered, and although not significant, they were higher in the blastocyst transfer group (42.59 vs 28.12%). Our conclusion supports considering single embryo transfer (SET) policy, even in cleavage stage in patients younger than 39 years with at least four zygotes. TRIAL REGISTRATION: ClinicalTrials.gov registration number NCT02639000.

A new, simple, automatic vitrification device: preliminary results with murine and bovine oocytes and embryos.

PURPOSE: This paper reports the use of a novel automatic vitrification device (Sarah, Fertilesafe, Israel) for cryopreservation of oocytes and embryos. METHODS: Mice oocytes (n = 40) and embryos (8 cells, n = 35 and blastocysts, n = 165), bovine embryos (2PN, n = 35), and MII oocytes (n = 84) were vitrified using this automated device. A total of 42 (2 cells) mice embryos, 20 (2PN) bovine embryos, and 150 MII bovine oocytes were used as fresh controls and grown to blastocysts. Upon rewarming, all were assessed for viability, cleavage, blastocyst, and hatching rates. RESULTS: Ninety-five % (38/40) of the mice MII oocytes regained isotonic volumes and all (100%) the surviving were viable. Rewarmed 8-cell mice embryos had 95% (33/35) blastulation rate and 80% (28/35) hatched. Rewarmed mice blastocysts had 97% survival rate (160/165) and 81% (135/165) hatched. Fresh control mice embryos had 100% (42/42) blastulation and 73% (21/42) hatching rates. Bovine embryos' survival was 100% with 54% (19/35) cleavage and 9% (3/35) blastulation rate. Fresh control bovine embryos had 65% (13/20) cleavage and 20% (4/20) blastulation rate. Vitrified bovine oocytes had 100% survival (84/84), 73% (61/84) cleavage, and 7% (6/84) blastocysts' rates; fresh control had 83% (125/150) cleavage and 11% (17/150) blastocysts' rates. CONCLUSION: This novel automatic vitrification device is capable to produce high survival rates of oocytes and embryos. We anticipate that as the demand for vitrification of gametes, embryos, and reproductive tissues increases worldwide, the availability of an automated vitrification device will become indispensable for standardization, simplification, and reproducibility of the entire process.

Delayed childbearing and female ageing impair assisted reproductive technology outcome in survivors of male haematological cancers.

PURPOSE: To analyse the impact of female characteristics on assisted reproductive technology outcome among male haematological cancer survivors. METHODS: A retrospective analysis of 93 haematological cancer survivors attending our tertiary referral fertility centre between June 1998 and June 2017 for achieving fatherhood with assisted reproductive technology treatments. RESULTS: A progressive increase in the median female age was observed during the study period (32.2 years until the year 2007 and 36.9 years from the year 2012). Fifty-five out of 93 patients were treated with intracytoplasmic sperm injection (ICSI) (113 ovarian stimulations, 108 ICSI procedures). Cryopreserved ejaculated sperm was used in 28 couples, fresh sperm in 19, and thawed testicular sperm in 8 couples. Mean female age at ovarian stimulation was 37.0 ± 4.7 years. Twenty-six pregnancies resulted in a full-term birth (23% per started ovarian stimulation; 43.6% per couple) and 33 children were born. No significant differences were observed according to source of sperm (fresh, frozen, testicular) and multivariate analysis confirmed that maternal age was the only variable inversely related to the cumulative delivery rate, being five times lower (15.7%) when the female partner was ≥ 40 years (OR = 0.22, 95% CI 0.06-0.77) vs. 58.3% with younger women (p = 0.0037). CONCLUSIONS: Delayed childbearing and female ageing affect ICSI outcome in couples where the male is a survivor of haematological cancer. This topic should be discussed when counselling male cancer patients about fertility preservation.

Proteomic profile of maternal-aged blastocoel fluid suggests a novel role for ubiquitin system in blastocyst quality.

PURPOSE: The etiology of maternal aging, a common cause of female factor infertility and a rate-limiting step in vitro fertilization (IVF) success, remains still unclear. Proteomic changes responsible for the impaired successful pregnancy outcome after IVF with aged blastocysts have not been yet evaluated. The objective of this prospective study was to employ proteomic techniques and bioinformatic tools to enlight differences at the protein level in blastocoel fluid of aged and younger woman. METHODS: Protein composition of human blastocoel fluid isolated by micromanipulation from 46 blastocysts of women aged <37 years (group A) and 29 of women aged ≥37 years (group B) have been identified by a shotgun proteomic approach based on high-resolution nano-liquid chromatography electrospray-ionization-tandem mass spectrometry (nLC-ESI-MS/MS) using label free for the relative quantification of their expression levels. RESULTS: The proteomic analysis leads to the identification and quantification of 148 proteins; 132 and 116 proteins were identified in groups A and B, respectively. Interestingly, the identified proteins are mainly involved in processes aimed at fine tuning embryo implantation and development. Among the 100 proteins commonly expressed in both groups, 17 proteins are upregulated and 44 downregulated in group B compared to group A. Overall, the analysis identified 33 proteins, which were increased or present only in B while 76 were decreased in B or present only in A. CONCLUSIONS: Data revealed that maternal aging mainly affects blastocyst survival and implantation through unbalancing the equilibrium of the ubiquitin system known to play a crucial role in fine-tuning several aspects required to ensure successful pregnancy outcome.