BACKGROUND: The aim of the present study is to investigate cryopreservation of oocytes in patients refusing embryo cryopreservation for ethical reasons, patients from whom no sperm could be retrieved and patients with enough oocytes to yield a number of fresh and cryopreserved embryos to transfer. METHODS: A total of 2900 oocytes out of 6216 retrieved were cryopreserved in 286 patients undergoing 303 cycles. The reasons for cryopreservation were because no sperm was found in 16 cycles, for ethical or personal reasons in 80, and in 207 only supernumerary oocytes were frozen. In 159 cycles, the oocytes were thawed and the surviving metaphase II oocytes microinjected. RESULTS: A toal of 1087 oocytes were thawed, 760 (69.9%) survived and 687 were microinjected. We obtained 368 (53.5%) normally cleaved embryos, 331 were transferred and 37 were cryopreserved. One hundred and forty-five transfers (range 1–3 embryos/patient) were performed and 18 (12.4%) pregnancies were obtained. Twelve patients delivered 13 healthy children, and six first trimester abortions were observed (33.3%). CONCLUSION: Although a low implantation rate was observed and a higher abortion rate than in fresh cycles, our results show that in sibling oocytes, the process of cryopreservation apparently does not affect the fertilization and cleavage rate. In this group of patients, producing a large number of mature gametes, oocyte cryopreservation gives the couple extra chances to achieve a pregnancy within a single retrieval and is a good effort towards reducing the number of embryos cryopreserved and enhancing our experience in this new technology.

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))? 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. 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. 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. 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. 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. 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. 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. None. None.

Can a second round of biopsy, vitrification and chromosomal testing provide a valid diagnosis where the first attempt fails? 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. 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. 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. 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. 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. 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. It is important to re-biopsy and re-vitrify undiagnosed blastocysts since healthy live births can result from them. None. None.

Oxidative stress (OS) plays an essential role in male infertility aetiology by affecting sperm quality, function, and also the integrity of sperm DNA. The assessment of oxidative stress in semen may be an important tool to improve the evaluation of sperm reproductive capacity. The purpose of this study was the evaluation of any possible relation between the unbalance of oxidative stress caused by superoxide anion in the ejaculate with the presence of sperm DNA fragmentation and high concentration of round cells. 56 semen samples from males from couples suffering from infertility were evaluated according to World Health Organisation (WHO) 2010 guidelines. Oxidative stress levels from N1 (low) to N4 (high) were assessed in ejaculates using oxiSperm; DFI (sperm DNA fragmentation index) as assessed by the SCSA (Sperm Chromatin Structure Assay) was used for evaluation of sperm chromatin integrity. Our data show that high oxidative stress (N3-N4 levels) correlated positively with aDFI≥30%(P=0.0379)and round cells≥1.500.000/mL(P=0.0084). In conclusion, OS increases sperm DNA damage. Thus evaluation of semen OS extent of sperm DNA damage in infertile man could be useful to develop new therapeutic strategies and improve success of assisted reproduction techniques (ART).

Background: folliculogenesis is a strictly regulated process that may be affected by endocrine disrupting chemicals (EDCs) through sometimes not so clear molecular mechanisms. Methods: we conducted a multicentric observational study involving six fertility centers across Italy, prospectively recruiting 122 women attending a fertility treatment. Recruited women had age ≤42 years, and normal ovarian reserve. Blood and follicular fluid samples were taken for EDCs measurement using liquid chromatography tandem mass spectrometry and each woman completed an epidemiological questionnaire. Results: The main EDCs found were monobutyl phthalate (MBP) (median blood: 8.96 ng/mL, follicular fluid 6.43 ng/mL), monoethylhexyl phthalate (MEHP) (median blood: 9.16 ng/mL, follicular fluid 7.68 ng/mL) and bisphenol A (BPA) (median blood: 1.89 ng/mL, follicular fluid 1.86 ng/mL). We found that serum MBP concentration was significantly associated with the considered area (p < 0.001, adj. mean: 7.61 ng/mL, 14.40 ng/mL, 13.56 ng/mL; Area 1: Milan–Turin, Area 2: Rome–Naples; Area 3: Catania–Bari, respectively) but negatively with home plastic food packaging (p = 0.004). Follicular MBP was associated with irregular cycles (p = 0.019). No association was detected between EDCs and eating habits and other clinical and epidemiological features. Conclusions: This study represents the first Italian biomonitoring of plastic EDCs in follicular fluid, laying the basis for future prospective evaluation on oocyte quality before assisted reproduction techniques (ART).