Abstract Study question Does semen samples quality contribute to blastocyst diversity by correlating with the presence of DNA in the blastocoelic fluid (BF)? Summary answer Semen samples quality is associated with the detection of DNA in BFs from both euploid and aneuploid blastocysts What is known already DNA was detected by whole genome amplification (WGA) in BFs from expanded blastocysts dependently on the blastocyst chromosome condition (assessed by PGT-A on trophectoderm (TE) cells), being more frequent in aneuploid blastocysts. TE-euploid blastocysts with positive BF-WGA also seem to have less chances to implant compared to those with failed BF-WGA. The extrusion of abnormal cells from the embryo proper was proposed as a mechanism triggered by the mosaic embryo to eliminate abnormal cells. Severe male factor samples are at risk of causing embryo mosaicism. Possible association between semen quality and DNA in BFs has not yet been investigated. Study design, size, duration This retrospective cohort study included 115 patients (maternal age 37.9±4.1), which underwent PGT-A in the last three years. The aim of the study was to evaluate whether the detection of DNA in BFs was correlated with semen samples indices. Both TE-euploid and TE-aneuploid blastocysts were considered for the analysis and stratified according to the semen samples quality as normozoospermic (N), moderate oligoasthenoteratozoospermic (m-OAT) and severe OAT (s-OAT) following the 2021 WHO criteria. Participants/materials, setting, methods BF and trophectoderm (TE) biopsies were collected from high grade expanded blastocysts before vitrification, and submitted to WGA. Amplification after WGA was evaluated by loading an aliquot of the amplified product onto a 1.5% agarose gel. 24-chromosome analysis was performed on TE biopsies. To assess the impact of semen samples quality on the detection of DNA in BFs, a multiple logistic regression analysis was performed correcting for maternal age as a confounding factor. Main results and the role of chance The BF from 590 blastocysts (265 TE-euploid and 325 TE-aneuploid) were submitted to WGA. In TE-euploid blastocysts, 125 derived from N semen samples, 52 from m-OAT, and 88 from s-OAT. The incidence of positive BF-WGA was proportional to the severity of the male factor condition and occurred in 48.0% of blastocysts originated from N samples, in 55.8% of blastocysts derived from m-OAT, and in 60.2% of blastocysts from s-OAT. In TE-aneuploid blastocysts, 178 originated from N semen samples, 80 from m-OAT, and 67 from s-OAT. The incidence of positive BF-WGA showed an opposite trend compared with the TE-euploid group being detected in 64.0% of blastocysts originated from N samples, in 66.3% of blastocysts derived from m-OAT, and in 52.2% of blastocysts obtained from s-OAT. The relationship between semen quality and positive BF-DNA amplification was confirmed by a multiple logistic regression model setting the BF-WGA as the outcome variable. In the TE-euploid blastocyst cohort, s-OAT showed an odd ratio (OR) of 1.78 (95% CI: 1.02-3.14; p=0.045), whereas in the TE-aneuploid blastocyst group, s-OAT displayed an OR of 0.55 (95% CI: 0.31-0.98; p=0.043). Limitations, reasons for caution This is a retrospective cohort study including a limited number of cases where the different categories of sperm samples were not equally represented. Although the results of BF-WGA were evaluated per single blastocyst, they cannot be considered independent variables. Wider implications of the findings In s-OAT samples, abnormal cell elimination is especially active in TE-euploid blastocysts, whereas in TE-aneuploid blastocysts the efficiency of this process is reduced suggesting the difficulty of eliminating abnormal cells in an aneuploid background. BF-DNA amplification is a minimally invasive tool contributing additional knowledge to elucidate blastocyst diversity. Trial registration number None

Abstract Study question Is the presence or absence of DNA in the blastocoelic fluid detected by whole genomic amplification (WGA) a valid method to prioritize embryos for transfer? Summary answer Blastocysts with DNA in the BF have lower ongoing pregnancy rates compared with blastocysts without DNA, both in PGT-A and in conventional IVF cycles What is known already The detection of DNA in BFs from expanded blastocysts has been reported in different studies. After amplification, this DNA can be analyzed to provide information on the blastocyst chromosome condition, but the degree to which it is representative of the corresponding embryo ploidy is still controversial. The reason of this divergence could reside in several factors, including the different status of the studied embryos. A recent study comparing euploid and aneuploid blastocysts reported a significantly higher incidence of failed BF-DNA amplification in euploid blastocysts compared with aneuploid blastocysts suggesting an effect of the embryo ploidy condition on BF content Study design, size, duration This prospective study included 142 cycles with PGT-A (Group-1; 24-chromosome analysis was performed on trophectoderm (TE) biopsies) and 121 conventional IVF consecutive cycles (Group-2) treated in the last three years. In both groups, the BF was collected from expanded blastocysts before vitrification, and submitted to WGA. Single blastocyst transfers were performed by selecting blastocysts based on BF-WGA results giving priority to those with failed amplification. In Group-1, only TE-euploid blastocysts were transferred Participants/materials, setting, methods Patients in Group-1 had a maternal age higher than in Group-2 (36.8±3 vs 34.1±3.5 years). The same protocol of vitrification was used for all patients, and only expanded blastocysts of high grade were included in the study. Amplification after WGA was evaluated by loading an aliquot of the amplified product onto a 1.5% agarose gel. An ongoing pregnancy was defined as a pregnancy regularly ongoing beyond the 16th week of gestation Main results and the role of chance In Group-1, a total of 622 blastocysts underwent trophectoderm (TE) biopsy and 261 were euploid. The BF was retrieved from 237 euploid blastocysts and submitted to WGA. Amplification failure resulted in 98 BFs, whereas 139 BF gave a positive amplification. In all, 57 clinical pregnancies resulted, 53 of which were regularly ongoing. 61 transfers were performed with euploid blastocysts with failed BF-WGA, and 81 with positive BF-WGA. When looking at the transfer outcome, the ongoing pregnancy rate was significantly higher for euploid blastocysts with failed BF-WGA (31/61, 50.8%) when compared to those with positive BF-WGA results (22/81, 27.2%, p<0.01). In Group-2, there were 62 clinical pregnancies, 52 of which were ongoing. In relation to the BF-WGA results, the ongoing pregnancy rate showed the same trend of Group-1, and was 20% (52/121) for blastocysts with failed BF-WGA and 59.1% (42/71, p<0.001) for blastocysts with positive BF-WGA Limitations, reasons for caution This is a prospective cohort study. The results should be confirmed by a prospectively randomized study Wider implications of the findings The presence of DNA in the BF could be indicative of an abnormal embryos that is trying to reach a viable condition. Therefore, failure to detect DNA after BF amplification could represent an additional criterion to select viable embryos for transfer both in PGT-A and in conventional IVF cycles Trial registration number Not applicable

STUDY QUESTION: Is de novo segmental aneuploidy (SA) a biological event or an artifact that is erroneously interpreted as partial chromosome imbalance? SUMMARY ANSWER: The detection of de novo SA in sequential biopsies of preimplantation embryos supports the biological nature of SA. WHAT IS KNOWN ALREADY: Although some SAs are detected in oocytes and in blastocysts, the highest incidence is observed in cleavage-stage embryos. Based on these findings, we can postulate that the majority of cells affected by SAs are eliminated by apoptosis or that affected embryos mainly undergo developmental arrest. STUDY DESIGN, SIZE, DURATION: This retrospective study includes 342 preimplantation genetic testing for aneuploidy (PGT-A) cycles performed between January 2014 and December 2018. Chromosome analysis was performed on 331 oocytes, 886 cleavage-stage embryos and 570 blastocysts (n = 1787). From 268 expanded blastocysts, the blastocoelic fluid (BF) was also analyzed (resulting in 2025 samples in total). In cases of SAs involving loss or gain in excess of 15 Mb, embryos were not considered for transfer and sequential biopsies were performed at following stages. This resulted in 66 sets where the initial diagnosis of SAs (4 made in polar bodies, 25 in blastomeres and 37 in trophectoderm (TE) cells) was followed up. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 2082 samples (2025 + 27 whole embryos) were processed by whole genome amplification followed by array comparative genomic hybridization. MAIN RESULTS AND THE ROLE OF CHANCE: The incidence of SAs was 6.3% in oocytes, increased to 16.6% in cleavage-stage embryos (P < 0.001) and decreased to 11.2% in blastocysts (P < 0.025 versus oocytes; P < 0.01 versus cleavage-stage embryos). The highest incidence of SAs was found in BFs (26.1%, P < 0.001). The analysis of 66 sets of sequential biopsies revealed that the initial finding was confirmed in all following samples from 39 sets (59.1% full concordance). In 12 additional sets, SAs were detected in some samples while in others the interested chromosome had full aneuploidy (18.2%). In three more sets, there was a partial concordance with the initial diagnosis in some samples, but in all TE samples the interested chromosome was clearly euploid (4.5%). In the remaining 12 sets, the initial SA was not confirmed at any stage and the corresponding chromosomes were euploid (18.2% no concordance). The pattern of concordance was not affected by the number of SAs in the original biopsy (single, double or complex) or by the absence or presence of concomitant aneuploidies for full chromosomes. LIMITATIONS, REASONS FOR CAUTION: Chromosome analyses were performed on biopsies that might not be representative of the true constitution of the embryo itself due to the occurrence of mosaicism. WIDER IMPLICATIONS OF THE FINDINGS: The permanence of SAs throughout the following stages of embryo development in more than half of the analyzed sets suggests for this dataset a very early origin of this type of chromosome imbalance, either at meiosis or at the first mitotic divisions. Since SAs remained in full concordance with the initial diagnosis until the blastocyst stage, a corrective mechanism seems not to be in place. In the remaining cases, it is likely that, as for full chromosome aneuploidy, mosaicism derived from mitotic errors could have occurred. In following cell divisions, euploid cell lines could prevail preserving the embryo chances of implantation. Due to the scarcity of data available, the transfer of embryos with SAs should be strictly followed up to establish possible clinical consequences related to this condition.

Introduction Preimplantation genetic testing for monogenic diseases (PGT-M) is a clinical method developed to prevent the transmission of monogenic inherited disorders to the future offspring. The reproductive risk of carriers of single gene disorders depends on the typology of the disorder, with the probability of affected conceptions ranging from 25% in recessive or X-linked diseases to 50% in dominant diseases. Therefore, the possibility of having embryos suitable for transfer and the consequent chances of ongoing pregnancy are strictly related to the number of embryos available for the genetic analysis. The aim of this study was to evaluate how many embryos need to be analyzed for having one or more genetically transferrable embryos. Material and Methods 86 carriers of genetic disorders performed 99 cycles of genetic analysis between January 2010 and September 2018. They were divided into two groups according to the typology of the disorder transmission. Group 1 included recessive and X-linked diseases (58 patients, 70 cycles of analysis), while with dominant disorders were in Group 2 (28 patients, 29 cycles of analysis). Maternal age was comparable in the two groups. Following blastocyst biopsy, embryos were vitrified to complete the genetic analysis. Results Clinical results were the following. For both groups, the mean number of analyzed embryos to have genetically transferrable embryos are reported in the following table: Conclusions Based on the reported data, we define for each case the minimum number of analyzable embryos needed to have one or more transferrable embryo depending on the typology of genetic disorder. Therefore, we eventually advise patients to undergo further oocyte retrievals for the best clinical outcome.

This study included 173 young couples of proven fertility who had previously undergone preimplantation genetic screening for chromosomes X and Y for family balancing. Several months later, when the outcome of the pregnancies was already known, the blastomeres from the corresponding embryos transferred were reanalysed by fluorescence in-situ hybridization (FISH) for chromosomes 13, 16, 18, 21, 22 with the aim of investigating correlation with embryo viability and the level of FISH sensitivity (embryos confirmed to be euploid). According to the results, informative in 152 couples, the proportion of euploid embryos was significantly lower in 53 nonpregnant women when compared with 99 women with term pregnancy (49% versus 75% respectively, P < 0.001). In addition, in 21 nonpregnant patients, all embryos transferred were found to be chromosomally abnormal. The level of FISH sensitivity was calculated in the group of term pregnancies where the number of euploid embryos was expected to exceed or match with the number of babies born. The resulting false-negative rate was 4.0% per patient and 1.9% per embryo. These findings confirmed the limited prediction power of embryo morphology on implantation but also the relevance of chromosomal abnormalities in causing embryo demise.

Background: Transition metal ions, such as iron, can make electron donations to oxygen forming superoxide or hydrogen peroxide, which is further reduced to an extremely reactive hydroxyl radical that induces oxidative stress. The purpose of the present study was to design a system that could easily detect and reliably measure the ferrous oxidation associated to oxygen radical reactions in the sperm samples. Methods: A total of 64 sperm samples from 11 men who had normal semen parameters and proven fertility and 53 male partners of couple experiencing primary infertility, were included in the study. The semen samples from oligoasthenoteratozoospermic patients was divided on the basis of spermatic parameters into moderate, when the sperm concentration was ≥5 × 106/ml and in severe when the concentration was <5 × 106/ml. The evaluation of the ferrous oxidation was performed measuring the formation of iron complexes between ferric ions and thiocyanate anions by spectrofluorimetry. Results: The concentration of the ferric thiocyanate complex ions was significantly higher in pathological sperm samples (137.6 ± 10.8 μmol/l in moderate oligoasthenoteratozoospermic, 170.0 ± 25.4 μmol/l in severe oligoasthenoteratozoospermic and 155.4 ± 7.3 μmol/l in non-obtructive azoospermic men), when compared with both infertile noormozoospermic (92.4 ± 10.7 μmol/l) (P<0.015) and with samples from fertile men (76.3 ± 6.2 μmol/l) (P<0.005). No significant differences were found in the concentration of ferric thiocyanate complex among the different pathological groups when compared to each other and in infertile noormozoospermic patients when compared with the samples from men of proven fertility (P=0.168). Accordingly, an inverse correlation was found between the concentration of the ferric thiocyanate complex and total motility, progressive motility and morphology. Conclusions: This preliminary study shows that the method proposed detect quickly and reliably measures the ferrous oxidation associated to oxygen radical reactions in the sperm samples.

BACKGROUND:To estimate the incidence of aneuploidy in relation to patients' characteristics, the type of hormonal stimulation and their response to induction of multiple follicular growth, 4163 first polar bodies (PB1s) were analyzed.METHODS:Five hundred and forty four infertile couples underwent 706 assisted conception cycles (640 with poor prognosis indications and 66 controls) in which chromosomal analysis of PB1 for the chromosomes 13, 15, 16, 18, 21 and 22 was performed. Results were evaluated in a multivariate analysis.RESULTS:The proportion of normal oocytes was directly correlated (P < 0.01) with (i) the number of mature oocytes and (ii) the establishment of a clinical pregnancy; and inversely correlated (P < 0.01) with (i) female age, (ii) causes of female infertility (endometriosis, abortions, ovulatory factor), (iii) poor prognosis indications (female age, number of previous cycles, multiple poor prognosis indications), (iv) number of FSH units per oocyte and (v) number of FSH units per metaphase II oocyte. There was a weak significance of frequency (P < 0.05) between type of abnormality (originated by chromatid predivision, chromosome non-disjunction or combined mechanisms in the same oocyte) and groups of the studied variables, rather than to a specific abnormality or a specific chromosome.CONCLUSIONS:The type of infertility had a significant effect on errors derived from the first meiotic division, whose incidence was significantly higher in the presence of endometriosis or of an ovulatory factor, and in women that experienced repeated abortions. Each aneuploidy event was found to be dependent not on a specific variable, but on groups of variables. In addition, the tendency of chromosomal abnormalities to occur simultaneously implies that the deriving aneuploidies can be of any type.