Genetic findings in miscarriages and their relation to the number of previous miscarriages.

PURPOSE: Early pregnancy loss leads to a devastating situation for many couples. Genetic disorders found in the pregnancy tissue are a frequent cause of miscarriages. It is unclear whether maternal age or previous miscarriages are associated with a higher chromosomal anomaly rate. This study aimed to determine the cytogenetical distribution of chromosomal disorders in couples after one or more previous miscarriages as well as the influence of maternal age. METHODS: 406 fetal tissue samples obtained after spontaneous abortion between 2010 and 2014 were successfully karyotyped. This included 132 couples with at least two losses and 274 couples with sporadic miscarriage. Normal and abnormal karyotype rate was determined for age, parity, gravidity, gestational week and number of previous miscarriages by logistic regression analysis. RESULTS: 145 (35.71%) fetal tissue samples had a normal karyotype, and 261 (64.8%) did not. After adjusting for age, older patients have a statistically significantly higher probability of genetic disorders in the pregnancy tissue (p < 0.001, OR 1.064, 95% CI 1.03-1.11). With each additional year, the probability of finding chromosomal abnormalities in a miscarriage increased by 6.4%. Patients younger than 35 years have a lower probability of having chromosomal disorders in the aborted material after two or more miscarriages than after sporadic miscarriages (50.7 vs. 58.9%) (p = 0.014, OR 0.67, 95% CI 0.48-0.914). Nevertheless, the risk of embryonic chromosomal disorders in patients aged 35 and above increased from 75.5% in sporadic miscarriages to 82.4% after more than one pregnancy losses (p = 0.59, OR 1.14, 95% CI - 0.72 to 1.92). CONCLUSION: Chromosomal disorders found after one or more previous miscarriages are related to patients' age. Couples suffering two or more miscarriages should be further researched, especially in younger patients.

Mojave rattlesnakes (Crotalus scutulatus scutulatus) lacking the acidic subunit DNA sequence lack Mojave toxin in their venom.

The venom composition of Mojave rattlesnakes (Crotalus scutulatus scutulatus) differs in that some individuals have Mojave toxin and others do not. In order to understand the genetic basis for this difference, genomic DNA samples from Mojave rattlesnakes collected in Arizona, New Mexico, and Texas were analyzed for the presence of DNA sequences that relate to the acidic (Mta) and basic (Mtb) subunits of this toxin. DNA samples were subjected to PCR to amplify nucleotide sequences from second to fourth exons of the acidic and basic subunits. These nucleotide sequences were cloned and sequenced. The nucleotide sequences generated aligned exactly to previously published nucleotide sequences of Mojave toxin. All DNA samples analyzed generated product using the basic subunit primers, and aligned identically to the Mtb nucleotide sequence. However, only 11 out of the 14 samples generated a product with the acidic subunit primers. These 11 sequences aligned identically to the Mta nucleotide sequence. The venom from the three snakes whose DNA did not amplify with the acidic subunit primers were not recognized by antibodies to Mojave toxin. This suggests that snakes with venom lacking Mojave toxin also lack the productive nucleotide sequence for the acidic subunit in their DNA.

The Alcaligenes eutrophus hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase, is required for heme biosynthesis during anaerobic growth.

The insertion mutant HF231 of Alcaligenes eutrophus H16 failed to grow anaerobically on nitrate and nitrite. When grown under oxygen limitation, mutant HF231 specifically excreted coproporphyrin III, an intermediate of heme biosynthesis. With the help of a Tn5-labeled fragment, we identified and cloned the corresponding wild-type fragment. Sequence analysis of the mutant locus revealed an open reading frame consisting of 1,473 bp, predicting a protein of 491 amino acids that corresponds to a size of 54.2 kDa. In the non-coding upstream region, consensus elements that are indicative for binding sites of the anaerobic transcriptional regulator Fnr were identified. The deduced polypeptide showed extensive sequence similarity with various bacterial oxygen-independent coproporphyrinogen III oxidases designated HemN. HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to yield protoporphyrinogen IX. Anaerobic growth on nitrate and nitrite of mutant HF231 was restored by introducing the hemN gene of A. eutrophus or of Pseudomonas aeruginosa on a broad-host-range vector. Likewise, the A. eutrophus hemN complemented heme biosynthesis of a Salmonella typhimurium hemF/hemN double mutant during anaerobic and aerobic growth. Analysis of a transcriptional lacZ gene fusion showed that expression of hemN in A. eutrophus is nitrate-independent and repressed by oxygen.

Differences in fibrinolysis and complement inactivation by venom from different northern blacktailed rattlesnakes (Crotalus molossus molossus).

Venom from 72 different Crotalus molossus molossus rattlesnakes was examined for fibrinolysis and for their ability to inactivate human complement. The fibrinolytic activity of the venoms was variable, but smaller (younger) snakes had less fibrinolytic activity than larger (older) snakes. Major differences between the venoms was detected by isoelectric focusing, and reflected in the number and pI of the proteins with fibrinolytic activity. Of the 72 venoms tested, ten had no effect and three had low activity on complement. The rest of the venoms strongly inactivated complement. The snakes with no activity on complement measured 55 cm or less in length, except for one snake which measured 53 cm and completely inactivated complement. Two larger snakes (76 and 84 cm) had a reduced complement-inactivating activity. Some venoms strongly hydrolyzed C2, whereas others had mild or no effect on this complement component. The attack on C3 was variable: some had no effect on C3, while other venoms produced a 125,000 mol. wt protein, which was recognized by antibodies to C3. Only mild hydrolysis of C4 was evident in serum treated with some venoms. No relationship was evident between the venom properties of this species and geographical distribution. Venom variability is an important clinical reality, and is an important consideration when attempting to isolate proteases from this snake species for further study.

Detection of alkaline phosphatase in venom by blotting methods.

Venom alkaline phosphatase was detected using a blotting method following electrophoresis. The enzyme gave strong reactions in some venoms, but was absent in other venoms, some within the same species. The mol. wt of the enzyme is close to 100,000 and its pI is between 3.6 and 4.8. The enzyme was inactivated by EDTA and 2-mercaptoethanol, and lost activity by freezing and thawing. Endogenous venom alkaline phosphatase can interfere with alkaline phosphatase-based detection methods. Pre-screening for endogenous venom alkaline phosphatase is recommended prior to using alkaline phosphatase-based detection methods when studying snake venom.

Hemorrhagic and Mojave toxins in the venoms of the offspring of two Mojave rattlesnakes (Crotalus scutulatus scutulatus).

1. The venoms of two Mojave rattlesnakes and those of their offsprings were analyzed for Mojave toxin and hemorrhagic toxin. 2. The venom of one female, collected in Pima County, Arizona, and the venoms of her six offspring contained hemorrhagic toxin but not Mojave toxin (venom B). 3. The venom of the second female, captured in El Paso County, Texas, contained both toxins (A+B venom). Of her 10 offspring, five contained venom with both toxins, two had hemorrhagic toxin only, and three contained neither toxin. 4. Venoms that caused hemorrhage also inactivated complement. A pool of the venoms of the venom B offspring was less toxic than adult pooled venom A.

Variation in the antigenic characteristics of venom from the Mojave rattlesnake (Crotalus scutulatus scutulatus).

Venoms from 31 specimens of the Mojave rattlesnake (Crotalus scutulatus scutulatus) were examined to further characterize reported differences among venoms of this species. Twenty-two venoms were recognized by a monoclonal antibody to Mojave toxin, CSS12. Nine venoms were recognized by CA-P-8, a monoclonal antibody produced against the hemorrhagic venom of C. atrox. Seven of these produced strong hemorrhage in mice and were also recognized by polyclonal antibodies (anti-F5) produced against a fraction of Mojave rattlesnake venom that inactivates serum complement. Fractionated venom revealed that CA-P-8 and anti-F5 recognized different proteins. Two of the venoms recognized by CA-P-8 were not recognized by anti-F5 and produced minimal hemorrhage in mice. This suggests that more than one factor may be necessary to induce strong hemorrhage.