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.

Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix.

The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.

Implication of the oligomeric state of the N-terminal PTX3 domain in cumulus matrix assembly.

Pentraxin 3 (PTX3) plays a key role in the formation of the hyaluronan-rich matrix of the cumulus oophorus surrounding ovulated eggs that is required for successful fertilization and female fertility. PTX3 is a multimeric protein consisting of eight identical protomers held together by a combination of non-covalent interactions and disulfide bonds. Recent findings suggest that the oligomeric status of PTX3 is important for stabilizing the cumulus matrix. Because the role of PTX3 in the cumulus resides in the unique N-terminal sequence of the protomer, we investigated further this issue by testing the ability of distinct Cys/Ser mutants of recombinant N-terminal region of PTX3 (N(_)PTX3) with different oligomeric arrangement to promote in vitro normal expansion in cumuli from Ptx3-null mice. Here we report that the dimer of the N(_)PTX3 is unable to rescue cumulus matrix organization, and that the tetrameric assembly of the protein is the minimal oligomeric state required for accomplishing this function. We have previously demonstrated that PTX3 binds to HCs of IαI and TSG-6, which are essential for cumulus matrix formation and able to interact with hyaluronan. Interestingly, here we show by solid-phase binding experiments that the dimer of the N(_)PTX3 retains the ability to bind to both IαI and TSG-6, suggesting that the octameric structure of PTX3 provides multiple binding sites for each of these ligands. These findings support the hypothesis that PTX3 contributes to cumulus matrix organization by cross-linking HA polymers through interactions with multiple HCs of IαI and/or TSG-6. The N-terminal PTX3 tetrameric oligomerization was recently reported to be also required for recognition and inhibition of FGF2. Given that this growth factor has been detected in the mammalian preovulatory follicle, we wondered whether FGF2 negatively influences cumulus expansion and PTX3 may also serve in vivo to antagonize its activity. We found that a molar excess of FGF2, above PTX3 binding capacity, does not affect in vitro cumulus matrix formation thus ruling out this possibility. In conclusion, the data strength the view that PTX3 acts as a nodal molecule in cross-linking HA in the matrix.

Molecular biomonitoring of a population of nurses handling antineoplastic drugs.

Many antineoplastic drugs have been found to have carcinogenic, mutagenic and teratogenic activity and so hospital personnel handling these substances are potentially exposed to health risk. Understanding this risk derived from protracted occupational exposure has great relevance even if the workers normally adopt individual and environmental protective measures. To address this question we have studied the presence of DNA and chromosome damage in a population of nurses employed in Italian oncology units and in matched controls. We used the comet assay to evidence the presence of DNA strand breaks, due to both acute and chronic exposure, and the micronucleus (MN) test, which is a measure of clastogenic and aneugenic events. Furthermore, since the individual response to the exogenous insults may be genetically determined, we studied the possible influence of single nucleotide polymorphism in XRCC1 and XRCC3 DNA repair genes on induced genetic damage. We also considered the effects of confounding factors like smoking, age and gender. The results indicated that the exposed subjects had significantly high levels of genetic damage. Age and gender were associated with increased values in MN, both in control and in exposed groups; the smoking habit affects MN frequency in controls, but not in workers. Furthermore we found that exposed subjects bearing at least one XRCC1 variant allele (399Gln) show higher values of MN. The present data provide the evidence to show that occupational exposure to antineoplastic drugs, even if in safety controlled conditions, represents a serious health risk. Furthermore we have shown that the presence of XRCC1 genetic polymorphism could contribute to increase the genetic damage in susceptible individuals who are occupationally exposed to dangerous substances.

Influence of glutathione S-transferase polymorphisms on genotoxic effects induced by tobacco smoke.

Genotoxicity of tobacco smoke has long been investigated and tobacco smoke is considered to be one of the principal human carcinogens. Although its role in DNA-damage induction and cancer development has been documented, the mechanisms by which this happens are not well understood. Many chemical constituents of tobacco smoke are enzymatically metabolized by phase-I and phase-II enzymes, but modifications in coding and regulating sequences of these genes could influence their ability to detoxify these compounds. In this work, we studied several enzymes involved in the metabolism of xenobiotics, viz. the glutathione S-transferases (GST) M1, T1, P1 and A1, with respect to their influence on the genotoxic effects induced by cigarette smoking. We assessed the genotoxic effects of tobacco smoke on peripheral blood lymphocytes of 72 healthy caucasians by use of the chromosomal aberration (CA) assay and the micronucleus (MN) test. Genotypes of GST M1, T1, P1 and A1 were determined by means of the polymerase chain reaction and methods based on restriction fragment length polymorphism (RFLP). We found that smoke and gender are the two variables that most influence the DNA damage. In particular, we observed that female smokers seem to be more sensitive than male smokers, having a significantly higher frequency of CAs. Moreover, a significant increase in frequency of micronuclei in bi-nucleated cells (BNMN) was found in smokers, but not in non-smokers. This increase seems to be influenced not only by age and gender, but also by genetic constitution. Subjects carrying GSTM1-null genotype seemed to have an higher susceptibility to DNA damage induced by tobacco smoke than GSTM1-positive ones. When considering a combination of GST genotypes, we found a lower BNMN frequency in subjects with GSTP1 variant allele plus GSTM1-positive genotypes, while the most damaged cells are found in subjects bearing GSTM1-null plus GSTP1-wild type. Our results suggest that investigation of the association between several gene polymorphisms and important endpoints of DNA damage could contribute to better understanding the role of gene-gene interaction.