Clinical outcomes following ICSI cycles using surgically recovered sperm and the impact of maternal age: 2004-2015 SART CORS registry.

PURPOSE: The aims of this study were (1) to evaluate clinical outcomes after ICSI cycles using surgically recovered sperm and (2) to assess the influence of maternal age on those outcomes. METHODS: A retrospective cohort study of 24,763 IVF cycles of fresh autologous oocytes and ICSI using surgically recovered sperm reported to the SART CORS database from 2004 to 2015. RESULTS AND CONCLUSIONS: Older women had significantly longer stimulation (p < 0.001), a lower number of oocytes retrieved (p < 0.001), a lower number of 2PN zygotes (p < 0.001), a lower chance of having a blastocyst transferred (p < 0.001), and a higher number of fresh embryos transferred (p < 0.001). There was no significant association between the number of 2PNs per oocyte retrieved and maternal age (p = 0.214). Both clinical pregnancy rates and live birth rates (LBR) decreased with advanced maternal age (p < 0.001). LBR ranged from 50.4% in women < 30 to 7.2% in women > 42 years, and for cleavage-stage transfers, the LBR ranged from 47.3% in women< 30 to 6.3% in women > 42 years. There were no differences in gestational age at delivery, proportion of term deliveries, preterm deliveries, neonatal birth weight < 2500 g, neonatal birth weight > 4000 g and average birthweight of neonates for singleton pregnancies according to age. For twin pregnancies, women < 30 years had significantly higher number of live births, term deliveries, and lower preterm deliveries than older women. There was a similar number of female (6051) and male neonates (5858; p = 0.2). Overall, pregnancy outcomes with ICSI using surgically recovered sperm are reassuring and comparable to those of ICSI with ejaculated sperm.

ICSI outcomes in men undergoing TESE for azoospermia and impact of maternal age.

This retrospective study compared clinical outcomes in men with obstructive and nonobstructive azoospermia after ICSI following testicular sperm extraction and the influence of maternal age. Fertilisation rates, embryo quality, pregnancy rates, miscarriage rates and live birth rates were evaluated. Men with obstructive azoospermia (OA) had significantly higher rates of diploid fertilisation and clinical pregnancy than men with nonobstructive azoospermia (NOA), but miscarriage rates and live birth rates were not significantly different. The higher rates of fertilisation, embryo quality and clinical pregnancy in men with OA were statistically significant when their female partners were <35 years but results were similar in both groups when female partners ≥35 years. Although the OA group had better overall quality embryos than the NOA group when maternal age was <35 years, embryologists can select the morphologically better embryos for transfer, eliminating the effect of embryo quality differences present in these two groups. Understanding more about factors that affect TESE/ICSI outcomes will not only help us predict patients' outcomes but it can help us educate and better counsel our patients.

Transcriptome of early embryonic invasion at implantation sites in a murine model.

Successful implantation relies on the interaction between a competent embryo and a receptive endometrium. The aim of the present study was to investigate genes differentially expressed in early invasive embryonic tissue versus decidual tissue in mice. Samples were obtained from the ectoplacental cone, the immediately surrounding deciduas and from deciduas from interimplantation sites. Microarray analysis showed that 817 genes were differentially expressed between extra-embryonic tissue and the surrounding decidua and that 360 genes were differentially expressed between the different deciduas, with a high representation of developmental processes. Genes differentially expressed in the maternal compartment included chemokines, lipoproteins, growth factors and transcription factors, whereas the embryonic invasive tissue expressed genes commonly observed in invasive tumour-like processes. These results provide information about genes involved in early embryonic invasion and the control exerted by the surrounding decidua. This information may be useful to find targets involved in pathologies associated with implantation failure and early pregnancy loss.

Milk fat globule epidermal growth factor 8 (MFG-E8): a novel protein in the mammalian endometrium with putative roles in implantation and placentation.

OBJECTIVES: MFG-E8 is a novel endometrial protein with conserved functions in tissue remodeling and angiogenesis in non-uterine tissues. Our aims were: 1. To examine the presence of MFG-E8 protein in the human endometrium during the window of implantation, in human endometrial cell lines, in human placental tissue at different gestational ages, and in murine implantation sites during early gestation; and 2. To study the regulation of MFG-E8 mRNA expression in mice implantation sites. STUDY DESIGN: MFG-E8 protein and its receptor integrin αvß3 were detected by immunostaining in human endometrial biopsies obtained from normal volunteers, in human endometrial cell lines (epithelial: Ishikawa and HEC-1A, stromal: HESC, and endothelial: HEEC), in human products of conception from all trimesters of gestation, and in murine implantation and inter-implantation sites dissected on days 5 and 8 post-coitus. MFG-E8 gene expression was assessed by RT-PCR. MAIN OUTCOME MEASURES: Immunohistochemical determination of MFG-E8 in endometrium and products of conception as well as relative MFG-E8 mRNA expression in mice implantation sites. RESULTS: MFG-E8 protein was present almost exclusively in the epithelial compartment of human endometrium. It was also expressed in the cytotrophoblasts and syncytiotrophoblasts outlining chorionic villi of the human placenta at all trimesters of gestation, and in murine implantation sites. MFG-E8 mRNA was significantly up-regulated in murine implantation sites and with increased gestational age. CONCLUSIONS: MFG-E8 expression in the endometrial epithelium as well as in chorionic villi suggests its possible role in endometrial reorganization during the receptive phase and in events related to normal pregnancy in mammals.

Impact of male age on the outcome of assisted reproductive technology cycles using donor oocytes.

This study assessed the influence of the age of the male partner on the outcome of oocyte donation cycles. A total of 408 couples participating in 519 consecutive anonymous oocyte donation cycles were examined. Main outcome measures were fertilization rate, embryo quality, clinical pregnancy, implantation, miscarriage and live birth rates, as well as the total reproductive potential, which estimates the outcome from fresh and cryopreserved-thawed embryo transfers. A total of 241 cycles resulted in clinical pregnancy (48.5% of transfers). The mean embryo score for transferred embryos (ESTE) was higher in cycles resulting in pregnancy (P=0.003). Semen volume (P<0.001), sperm motility (P<0.001) and fertilization rate (P=0.04) decreased significantly with advanced male age, which did not correlate with mean ESTE or implantation rate. Fertilization rate was the only predictor of ESTE (B=16.066, P=0.012), whereas inseminated/retrieved egg ratio was the only predictor of implantation rate (B=0.555, P=0.039). Pregnancy was only predicted by ESTE (Exp(B)=1.023, P<0.001), which also was the only predictor of live birth (Exp(B)=1.017, P=0.009). There was no predictor of miscarriage (47 cycles, 9.1%) identified. Although semen volume, sperm motility and fertilization rate decreased with advanced male age, embryo quality, clinical pregnancy, implantation, miscarriage and live birth rates were not affected.

Variations of the perforin gene in patients with multiple sclerosis.

Perforin is involved in cell-mediated cytotoxicity and mutations of its gene (PRF1) cause familial hemophagocytic lymphohistiocytosis (FLH2). PRF1 sequencing in 190 patients with multiple sclerosis and 268 controls detected two FLH2-associated variations (A91V, N252S) in both groups and six novel mutations (C999T, G1065A, G1428A, A1620G, G719A, C1069T) in patients. All together, carriers of these variations were more frequent in patients than in controls (phenotype frequency: 17 vs 9%, P=0.0166; odds ratio (OR)=2.06, 95% confidence interval (CI): 1.13-3.77). Although A91V was the most frequent variation and displayed a trend of association with multiple sclerosis (MS) in the first population of patients and controls (frequency of the 91V allele: 0.076 vs 0.043, P=0.044), we used it as a marker to confirm PRF1 involvement in MS and assessed its frequency in a second population of 966 patients and 1520 controls. Frequency of the 91V allele was significantly higher in patients than in controls also in the second population (0.075 vs 0.058%, P=0.019). In the combined cohorts of 1156 patients and 1788 controls, presence of the 91V allele in single or double dose conferred an OR=1.38 (95% CI=1.10-1.74). These data suggest that A91V and possibly other perforin variations indicate susceptibility to MS.

A sequence variation in the MOG gene is involved in multiple sclerosis susceptibility in Italy.

Several studies suggest that the histocompatibility complex (HLA) class I region harbours genes modulating multiple sclerosis (MS) susceptibility independently from the effect of class II alleles. A candidate gene in this region is MOG, encoding the myelin oligodendrocyte glycoprotein. A significant association with the missense variation V142L (rs2857766) was previously reported in a small sample of 50 Italian MS patients. We confirmed this result in two independent Italian sample sets consisting of 878 MS patients and 890 matched controls (P=6.6 x 10(-4)) and 246 trio families (P=1.5 x 10(-3)). The comparison of genotype frequencies suggested a dominant-protective effect of L142. In the combined sample sets L142 conferred an odds ratio (OR)=0.70 (95% confidence interval (CI): 0.60-0.82) that remained similar after accounting for HLA-DRB1(*)15 carrier status. The association with MOG V142L was still significant after conditioning for all DRB1 alleles (P=0.035). Eleven additional single nucleotide polymorphisms in the MOG gene (namely -1077T/C, -910T/C, -875A/G, -93T/C, S5S, Indel L22, V145I, +814C/T, +900A/G, +1024A/T, +1059C/T), two microsatellites in the MOG 5' flanking (MOGCA) and 3' untranslated (MOGTAAA) regions and four microsatellites in the HLA-class I region, from HLA-B to HFE, (namely MIB, D6S265, D6S1683 and D6S2239) were tested by transmission disequilibrium test in 199 trio families. None of these polymorphisms or of their haplotypic combinations showed a significant transmission distortion, in the absence of V142L. In conclusion, MOG V142L, or an untested variant in tight-linkage disequilibrium with it, is an independent MS susceptibility-modulating factor in the HLA class I region.

In search of candidate genes critically expressed in the human endometrium during the window of implantation.

BACKGROUND: In this prospective randomized blinded clinical trial, we examined gene expression profiles of the human endometrium during the early and mid-luteal phases of the natural cycle. METHODS: An endometrial biopsy was performed on day 16 (LH +3) or on day 21 (LH +8), followed by RNA extraction and microarray analysis using an Affymetrix HG-U95A microchip. Data analysis was carried out using pairwise multiple group comparison with the significance analysis of microarrays (SAM) software. RESULTS: With a false discovery rate of 0, the analysis revealed that 107 genes were significantly and differently expressed (> or =2-fold) during the early versus the mid-luteal phase of the cycle. Forty-five of these genes have not been previously linked to endometrial receptivity. Validation of the microarray data was accomplished using semiquantitative RT-PCR. We demonstrated the presence of estrogen and progesterone response elements (ERE and PRE) by analysis of the 5'-flanking regions of a subset of differentially regulated genes. CONCLUSIONS: Using a strict bioinformatics approach of microarray data, we demonstrated significant changes in candidate genes during the transition of the early to the mid-luteal phase of the human endometrium that may have functional significance for the opening and maintenance of the window of implantation.

Occurrence of a putative SCF ubiquitin ligase complex in Drosophila.

Many proteins are targeted to proteasome degradation by a family of E3 ubiquitin ligases, termed SCF complexes, that link substrate proteins to an E2 ubiquitin-conjugating enzyme. SCFs are composed of three core proteins-Skp1, Cdc53/Cull, Rbx1/Hrt1-and a substrate specific F-box protein. We have identified in Drosophila melanogaster the closest homologues to the human components of the SCF(betaTrCP) complex and the E2 ubiquitin-conjugating enzyme UbcH5. We show that putative Drosophila SCF core subunits dSkpA and dRbx1 both interact directly with dCu11 and the F-box protein Slmb. We also describe the direct interaction of the UbcH5 related protein UbcD1 with dCul1 and Slmb. In addition, a functional complementation test performed on a Saccharomyces cerevisiae Hrt1p-deficient mutant showed that Drosophila Rbx1 is able to restore the yeast cells viability. Our results suggest that dRbx1, dSkpA, dCullin1, and Slimb proteins are components of a Drosophila SCF complex that functions in combination with the ubiquitin conjugating enzyme UbcD1.

Inhibition of UDP-glucose: protein transglucosylase by a maize endosperm protein factor.

UDP-glucose: protein transglucosylase (UPTG, EC 2.4.1.112) catalyzes the first step of protein-bound alpha-glucan synthesis in potato tuber and developing maize endosperm. The presence of a non-dialyzable, heat labile protein responsible for low levels of UPTG activity in developing maize endosperm was investigated. UPTG activity in 5-day old maize seedlings and potato tuber solubilized preparations was also reduced by the endosperm preparation. FPLC-Mono Q column chromatography of developing maize endosperm was effective in separating the inhibitor protein (IP) from UPTG. After gel filtration on Superose 12, IP yielded a major polypeptide of about 80 kDa on SDS-PAGE. IP was purified by gel filtration on Superose 12 and preparative SDS-PAGE, and specific antibodies were prepared. Polyclonal antibodies reacted specifically with an 80 kDa polypeptide of developing maize endosperm on Western blot. They also recognized a similar band in 5-day old maize seedlings, but not in potato tubers. The identification of a factor that regulates the level of UPTG activity in developing maize endosperm may help to elucidate the functional role of the enzyme in the initiation of starch synthesis during seed development.

Sequence-specific DNA modification in Acetobacter xylinum.

Two cryptic plasmids have been discovered in Acetobacter xylinum B42 and in its derivative PEA-1, a cellulose defective mutant. These two plasmids were designated pAX1 and pAX2 (50 and 105 kb in size, respectively). A restriction map was constructed for pAX1. Attempts to cure these plasmids were unsuccessful. Enzyme restriction analysis showed that these plasmids contain protected EcoRI and ApoI sites. Using Southern blot and hybridization techniques, the protection was extended to chromosomal DNA. Enzyme restriction analysis of several plasmids, from different origins and containing different incompatibility groups, isolated from strain PEA-1 also showed EcoRI and ApoI protection. The presence of modifications on specific sequences was not found in A. xylinum 8747. These results strongly suggest the presence of a modification system in A. xylinum B42 that recognizes the tetranucleotide 5'-AATT.

Investigation of some biochemical and functional effects of cryopreservation of human spermatozoa using an automated freezing-quick-thawing method.

The objective of the present studies was to assess the functional integrity of the sperm plasma membrane and metabolic and motility characteristics of the recovered motile fraction of human spermatozoa subjected to an automated freezing/quick-thawing method. Sperm membrane features examined included progesterone-induced changes in intracellular levels of calcium ([Ca2+]i), as measured by the fluorescent fura-2 indicator, and the tight binding of spermatozoa to homologous zonae pellucidae as assessed by the hemizona assay (HZA). Basal [Ca2+]i intracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels determined using chemiluminescence with luciferin-luciferase, and motility parameters determined using a computer-aided semen analyser (CASA) were studied concomitantly as an expression of metabolic/functional status. Ejaculates from fertile men (donors) were evaluated after swim-up separation of the motile fraction in both fresh and cryopreserved-thawed samples, and fractions of each ejaculate (fresh and frozen-thawed) were subjected to parallel measurements of the same parameters at the same time frame. Basal and progesterone-induced increase in [Ca2+]i, and ATP levels (up to 24 h) were similar in fresh and frozen-thawed samples. HZA results showed a modest (26%) although significant (p = 0.008) decrease in binding in frozen-thawed samples. The ratios of ATP/ADP in fresh and frozen-thawed samples were also found to be similar. Although post-thaw sperm motility was significantly lower than that of the fresh samples, comparison of the results indicated that the method was capable of preserving > 65% of motile spermatozoa in almost all of the samples cryopreserved. Additionally, the swim-up rescued a motile fraction in the frozen-thawed samples that was not significantly impaired with regard to motility, mean linear velocity or linearity as compared to the fresh fractions in the first 4 h. Our results show that this automated freezing-quick-thawing method results in a small reduction in sperm-zona binding capacity, and that the time-dependent decline in motility parameters observed for both fresh and cryopreserved-thawed samples cannot be related to ATP deficiency under the conditions of our experiments. These in-vitro results are coincident with the maintenance of fertilizing capacity for donor spermatozoa in the in-vitro fertilization (IVF) setting.

[Multiple resistance to heavy metals and antibiotics in bacteria isolated from the Riachuelo]. / Resistencia múltiple a metales pesados y a antibióticos en bacterias aisladas del riachuelo.

Riachuelo is a heavy contaminated course of water, partially surrounding Buenos Aires city. The presence of bacteria resistant to antibiotics and heavy metals was studied. Among the isolated Gram positive colonies, 65, 59 and 48% were resistant to 60 micrograms/ml of Pb++, Zn++ and Cd++, respectively, and 20% grew in the presence of 50 micrograms/6ml gentamicin. Most of these microorganisms belonged to the order Actinomycetales. Accordingly, high percentages of resistance were detected (among the 11 Gram negative isolates (Enterobacteriaceae and Pseudomonaceae), although only one isolate was gentamicin resistant. Four Gram negative isolates also showed a broad spectrum of resistance to tetracycline, erythromyci, ampicillin, amikacin, chloramphenicol and gentamicin.

Validation of the hemizona assay (HZA) in a monkey model. II. Kinetics of binding and influence of cryopreserved-thawed spermatozoa.

We compared fresh and frozen-thawed cynomolgus monkey spermatozoa tight binding to the zona pellucida under hemizona assay (HZA) conditions. Monkey oocytes were recovered after superovulation and stored in salt solution. Matching hemizonae were obtained by micromanipulation. Semen, obtained by electroejaculation, was used fresh or was cyropreserved, thawed, and washed by swim-up separation. At the standard initial dilution of 500,000 motile sperm/ml (or 5 x 10(4) motile sperm/hemizona), binding was significantly higher for fresh sperm (P = 0.00004). For frozen-thawed samples, there was a linear increase in the number of tightly bound sperm with increasing sperm concentration (r = 0.95). At 1.5 x 10(6) motile sperm/hemizona, binding of frozen-thawed spermatozoa was similar to that of fresh at a standard concentration. Kinetic studies showed peak binding at 1 hr of gametes coincubation. We conclude that, in this monkey model, the HZA is a valuable bioassay for evaluation of sperm binding to the zona pellucida, the initial requisite for fertilization and embryo development.

Resistencia múltiple a metales pesados y a antibióticos en bacterias aisladas del riachuelo. / [Multiple resistance to heavy metals and antibiotics in bacteria isolated from the Riachuelo]

Riachuelo is a heavy contaminated course of water, partially surrounding Buenos Aires city. The presence of bacteria resistant to antibiotics and heavy metals was studied. Among the isolated Gram positive colonies, 65, 59 and 48

were resistant to 60 micrograms/ml of Pb++, Zn++ and Cd++, respectively, and 20

grew in the presence of 50 micrograms/6ml gentamicin. Most of these microorganisms belonged to the order Actinomycetales. Accordingly, high percentages of resistance were detected (among the 11 Gram negative isolates (Enterobacteriaceae and Pseudomonaceae), although only one isolate was gentamicin resistant. Four Gram negative isolates also showed a broad spectrum of resistance to tetracycline, erythromyci, ampicillin, amikacin, chloramphenicol and gentamicin.

Resistencia múltiple a metales pesados y a antibióticos en bacterias aisladas del riachuelo / [Multiple resistance to heavy metals and antibiotics in bacteria isolated from the Riachuelo]

Riachuelo is a heavy contaminated course of water, partially surrounding Buenos Aires city. The presence of bacteria resistant to antibiotics and heavy metals was studied. Among the isolated Gram positive colonies, 65, 59 and 48

were resistant to 60 micrograms/ml of Pb++, Zn++ and Cd++, respectively, and 20

grew in the presence of 50 micrograms/6ml gentamicin. Most of these microorganisms belonged to the order Actinomycetales. Accordingly, high percentages of resistance were detected (among the 11 Gram negative isolates (Enterobacteriaceae and Pseudomonaceae), although only one isolate was gentamicin resistant. Four Gram negative isolates also showed a broad spectrum of resistance to tetracycline, erythromyci, ampicillin, amikacin, chloramphenicol and gentamicin.

The YAG laser used in micromanipulation to transect the zona pellucida of hamster oocytes.

PROBLEM: Since there has been no reported use of the YAG laser to micromanipulate oocytes, our purpose was to study whether (1) a YAG laser could be used to open the zona pellucida of hamster oocytes; (2) human sperm could reach the ooplasm and (3) under sperm penetration assay conditions, sperm would bind and penetrate the ooplasm. RESULTS: A YAG 100 laser was used at 10 W and 0.4-sec pulse width to open eight of eight ooplasm oocytes. The opening in the zonae was 0.25 to 1.0 rad (10 to 40 microns). For the initial eight oocytes and two parallel controls, the coarse appearance of the ooplasm was unchanged after 3 days. Next, in 11 of 12 manipulated oocytes, the sperm clustered at the opening of the zona. When 16 more oocytes were opened and exposed to sperm in sperm penetration assay conditions, each ooplasm bound sperm. There was no penetration noted. Each manipulation time was < 1 min. To clarify the laser effect, oocytes were exposed to laser energy then utilized as the interactive surface in the sperm penetration assay. It was found that only 20% bound sperm with no penetration. CONCLUSION: While the time factor compares favourably with other methods of zona opening, further study needs to be performed to minimize effect to the exposed oocyte.

Cryopreserved/thawed semen for in vitro fertilization: results from fertile donors and infertile patients.

We evaluated the in vitro fertilization (IVF) outcome in 54 cycles using cryopreserved/thawed semen from fertile donors. Controls were other IVF patients matched by time frame, female age, stimulation protocol, number of pre-embryos transferred, and absence of a male factor using freshly ejaculated normal semen samples. In the study group and controls, respectively, post-thaw swim-up motility was 83.1% and 89.5%; fertilization rate of preovulatory oocytes (91.8%, 95.7%) and ongoing pregnancy rate (PR) per transfer (21.1%, 25.0%) were similar. The excellent fertilization rate with frozen/thawed semen was achieved through high-concentration insemination (0.5 x 10(6) motile sperm/mL). With use of frozen/thawed samples from infertile men (normal and subfertile samples), PR was similar but fertilization rate was lower. Cryopreserved semen is a valuable option for infertile couples in IVF therapy.