PCI-24781 can improve in vitro and in vivo developmental capacity of pig somatic cell nuclear transfer embryos

Objective

To examine the effect of PCI-24781 (abexinostat) on the blastocyst formation rate in pig somatic cell nuclear transferred (SCNT) embryos and acetylation levels of the histone H3 lysine 9 and histone H4 lysine 12.

Results

Treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos, in comparison to the control group (25.3 vs. 10.5 %, P < 0.05). Furthermore, PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12. TUNEL assay and Hoechst 33342 staining revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos. Also, PCI-24781-treated embryos were transferred into three surrogate sows, one of whom became pregnant and two fetuses developed.

Conclusion

PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.

     

RepSox improves viability and regulates gene expression in rhesus monkey–pig interspecies cloned embryos

Objective

To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey–pig interspecies somatic cell nuclear transfer (iSCNT) embryos.

Results

Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p < 0.05), but not of Sox2 compared with untreated embryos at the 2–4-cell stage. Expression of the anti-apoptotic gene, Bcl2, and the pro-apoptotic gene Bax was also affected at the 2–4-cell stage. RepSox treatment also increased the immunostaining intensity of Oct4 at the blastocyst stage (p < 0.05). Although the blastocyst developmental rate was higher in the group treated with 25 µM RepSox for 24 h than in the untreated control group (2.4 vs. 1.2%, p > 0.05), this was not significant.

Conclusion

RepSox can improve the developmental potential of rhesus monkey–pig iSCNT embryos by regulating the expression of pluripotency-related genes.

     

The developmental competence of oocytes parthenogenetically activated by an electric pulse and anisomycin treatment

Objective

The aim of this study was to investigate the developmental competence of oocytes parthenogenetically activated by an electric pulse (EP) and treated with anisomycin and to determine whether this method is applicable to somatic cell nuclear transfer (SCNT).

Results

Embryos derived from porcine oocytes parthenogenetically activated by an EP and treatment with 0.01 µg/mL anisomycin had a significantly improved in vitro developmental capacity. Furthermore, 66.6% of blastocysts derived from these embryos had a diploid karyotype. The blastocyst formation rate of cloned embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 0.01 µg/mL anisomycin for 4 h. The level of maturation-promoting factor was significantly decreased in oocytes activated by an EP and treated with anisomycin. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR.

Conclusion

Our results demonstrate that porcine oocyte activation via an EP in combination with anisomycin treatment can lead to a high blastocyst formation rate in parthenogenetic activation and SCNT experiments.

     

Fusion expression of cecropin B-like antibacterial peptide in <Emphasis Type="Italic">Pichia</Emphasis> GS115 and its antibacterial mechanism

Objectives

To establish an efficient expression system for a fusion protein of glutathione S-transferase and cecropin B (GST-CB) and to clarify the antibacterial mechanism of CB.

Results

The optimal incubation time and methanol concentration for induced expression of CB were 36 h and 1 % w/v, respectively. The yield of GST-CB was 2.2 g/l. The minimum inhibitory concentrations of GST-CB towards Staphylococcus aureus subsp. saprophyticus (ATCC 15305) and Escherichia coli strain CFT073 were 250 and 125 μg/ml, respectively. Notably, mutations of proline 24 (P24) in CB produced a polypeptide without antimicrobial activity.

Conclusion

The fusion protein GST-CB, which has a broad spectrum antimicrobial activity, can be abundantly expressed in Pichia pastoris GS115, and P24 may be an important amino acid for the antimicrobial activity of GST-CB.

     

Noninvasive characterization of metabolites secreted in culture media by bovine embryos during in vitro production

Introduction

Secreted molecules could be correlated with the potential of embryonic development. The development of new technologies, such as mass spectrometry (MS), has enabled analyzes in culture medium to favor the determination of embryos viability in order to improve embryo selection.

Objectives

To perform a non-invasive characterization of the secretome of in vitro produced embryos with different kinetics of cleavage and in different stages of development to obtain specific patterns based on embryonic phenotype through MALDI–TOF–MS.

Methods

Bovine embryos were produced in vitro by standard protocols. The zygotes were transferred to individual culture medium and divided into two groups: Fast [4 cells-22 hours past the beginning of culture (hpc)] and Slow (2 cells-22 hpc). Culture media drops were collected at 22, 96 and 168 hpc. Analysis of embryonic secretome was made by MALDI–TOF–MS after extractions of the metabolites. Spectra were acquired in positive ionization mode. Univariate (Fold-change) and multivariate (Partial Least Squares Discriminants Analysis) analyses were performed by the online software Metaboanalyst.

Results

It was demonstrated that embryos with different kinetics have different spectrometric profiles during embryonic development. Moreover, secreted molecules in each developmental stage are differentially represented in embryos with different kinetics, and are related to specific pathways such as lipid and amino acids metabolism and cell proliferation.

Conclusion

We propose that the analysis of culture media by MALDI–TOF–MS can be used for qualitative characterization of bovine embryos, allowing the identification of key molecules during in vitro culture.

     

Corneal Collagen Cross-Linking for the Management of Mycotic Keratitis

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.

     

A novel method for single-grain-based metabolic profiling of Arabidopsis seed

Introduction

In plant metabolomics, metabolite contents are often normalized by sample weight. However, accurate weighing of very small samples, such as individual Arabidopsis thaliana seeds (approximately 20 µg), is difficult, which may lead to irreproducible results.

Objectives

We aimed to establish alternative normalization methods for seed-grain-based comparative metabolomics of A. thaliana.

Methods

Arabidopsis thaliana seeds were assumed to have a prolate spheroid shape. Using a microscope image of each seed, the lengths of major and minor axes were measured by fitting a projected 2-dimensional shape of each seed as an ellipse. Metabolic profiles of individual diploid or tetraploid A. thaliana seeds were measured by our highly sensitive protocol (“widely targeted metabolomics”) that uses liquid chromatography coupled with tandem quadrupole mass spectrometry. Mass spectrometric analysis of 1 µL of solution extract identified more than 100 metabolites. The data were normalized by various seed-size measures, including seed volume (single-grain-based analysis). For comparison, metabolites were extracted from 4 mg of diploid and tetraploid A. thaliana seeds and their metabolic profiles were analyzed by normalization of weight (weight-based analysis).

Results

A small number of metabolites showed statistically significant differences in the single-grain-based analysis compared to weight-based analysis. A total of 17 metabolites showed statistically different accumulation between ploidy types with similar fold changes in both analyses.

Conclusion

Seed-size measures obtained by microscopic imaging were useful for data normalization. Single-grain-based analysis enables evaluation of metabolism of each seed and elucidates the metabolic profiles of precious bioresources by using small amounts of samples.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

The effect of kidney function on the urate lowering effect and safety of increasing allopurinol above doses based on creatinine clearance: a post hoc analysis of a randomized controlled trial

Background

The use of allopurinol in people with chronic kidney disease (CKD) remains one of the most controversial areas in gout management. The aim of this study was to determine the effect of baseline kidney function on safety and efficacy of allopurinol dose escalation to achieve serum urate (SU) <6 mg/dl.

Methods

We undertook a post hoc analysis of a 24-month allopurinol dose escalation treat-to-target SU randomized controlled trial, in which 183 people with gout were randomized to continue current dose allopurinol for 12 months and then enter the dose escalation phase or to begin allopurinol dose escalation immediately. Allopurinol was increased monthly until SU was <6 mg/dl. The effect of baseline kidney function on urate lowering and adverse effects was investigated.

Results

Irrespective of randomization, there was no difference in the percentage of those with creatinine clearance (CrCL) <30 ml/min who achieved SU <6 mg/dl at the final visit compared to those with CrCL ≥30 to <60 ml/min and those with CrCL ≥60 ml/min, with percentages of 64.3% vs. 76.4% vs. 75.0%, respectively (p?=?0.65). The mean allopurinol dose at month 24 was significantly lower in those with CrCL <30 ml/min as compared to those with CrCL ≥30 to <60 ml/min or CrCL ≥60 ml/min (mean (SD) 250 (43), 365 (22), and 460 (19) mg/day, respectively (p?<?0.001)). Adverse events were similar among groups.

Conclusions

Allopurinol is effective at lowering urate even though and accepting that there were small numbers of participants with CrCL <30 ml/min, these data indicate that allopurinol dose escalation to target SU is safe in people with severe CKD. The dose required to achieve target urate is higher in those with better kidney function.

Trial registration

Australian and New Zealand Clinical trials Registry, ACTRN12611000845932. Registered on 10 August 2011.

     

Does centrifugation matter? Centrifugal force and spinning time alter the plasma metabolome

Background

Centrifugation is an indispensable procedure for plasma sample preparation, but applied conditions can vary between labs.

Aim

Determine whether routinely used plasma centrifugation protocols (1500×g 10 min; 3000×g 5 min) influence non-targeted metabolomic analyses.

Methods

Nuclear magnetic resonance spectroscopy (NMR) and High Resolution Mass Spectrometry (HRMS) data were evaluated with sparse partial least squares discriminant analyses and compared with cell count measurements.

Results

Besides significant differences in platelet count, we identified substantial alterations in NMR and HRMS data related to the different centrifugation protocols.

Conclusion

Already minor differences in plasma centrifugation can significantly influence metabolomic patterns and potentially bias metabolomics studies.

     

Effects of sex on response of the bovine preimplantation embryo to insulin-like growth factor 1, activin A,and WNT7A

Background

Alterations in maternal environment can sometimes affect embryonic development in a sexually-dimorphic manner. The objective was to determine whether preimplantation bovine embryos respond to three maternally-derived cell signaling molecules in a sex-dependent manner.

Results

Actions of three embryokines known to increase competence of bovine embryos to develop to the blastocyst stage, insulin-like growth factor 1 (IGF1), activin A, and WNT member 7A (WNT7A), were evaluated for actions on embryos produced in vitro with X- or Y- sorted semen from the same bull. Each embryokine was tested in embryos produced by in vitro fertilization of groups of oocytes with either pooled sperm from two bulls or with sperm from individual bulls. Embryos were treated with IGF1, activin A, or WNT7A on day 5 of culture. All three embryokines increased the proportion of cleaved zygotes that developed to the blastocyst stage and the effect was similar for female and male embryos. As an additional test of sexual dimorphism, effects of IGF1 on blastocyst expression of a total of 127 genes were determined by RT-qPCR using the Fluidigm Delta Gene assay. Expression of 18 genes was affected by sex, expression of 4 genes was affected by IGF1 and expression of 3 genes was affected by the IGF1 by sex interaction.

Conclusion

Sex did not alter how IGF1, activin A or WNT7A altered developmental competence to the blastocyst stage. Thus, sex-dependent differences in regulation of developmental competence of embryos by maternal regulatory signals is not a general phenomenon. The fact that sex altered how IGF1 regulates gene expression is indicative that there could be sexual dimorphism in embryokine regulation of some aspects of embryonic function other than developmental potential to become a blastocyst.

     

Minimal variation of the plasma lipidome after delayed processing of neonatal cord blood

Background

Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing.

Method

Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy.

Results

Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased.

Conclusion

Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.

     

Effect of day 3 embryo morphometrics and morphokinetics on survival and implantation after slow freezing-thawing and after vitrification-warming: a retrospective cohort study

Background

Morphometric and morphokinetic evaluation of in vitro cultured human embryos allows evaluation without time restriction and reduces intra- and inter-observer variability. Even though these technologies have been reported to improve the quality of cleavage stage embryo evaluation during fresh culture, possible advantages in the evaluation of cryopreserved embryos have been scarcely explored. This study aims to compare morphometric and morphokinetic parameters between slow frozen and vitrified embryos and to determine their relationship to embryo survival and implantation rate (IR) after thawing/warming.

Methods

During fresh culture, morphometric characteristics (Total Cell Volume (TCV), symmetry, fragmentation and number of blastomeres) were measured in 286 thawed/warmed embryos. Likewise, after thawing/warming, similar morphometric characteristics were measured in 135 survived embryos. Moreover, morphokinetic parameters (time to mitosis resumption and time to compaction) were measured in 90 embryos after thawing/warming. Then, using linear regression, we investigated the differences between vitrified and slow frozen embryos and the relation of the measured characteristics to embryo survival and IR. Statistical corrections were applied to account for data clustering and for multiple testing.

Results

Vitrified embryos resume mitosis and start compaction significantly earlier than slow frozen embryos. Mitosis resumption rate was 82% for vitrified and 63% for slow frozen embryos and median time to mitosis resumption was 7.6 h and 13.1 h (p = 0.02), respectively. Compaction rate was 62% in vitrified and only 23% in slow frozen embryos. Median time to compaction was 18.1 h for vitrified embryos but, for slow frozen could not be computed since less than half of the slow frozen embryos reached compaction (p = 0.0001). Moreover, intact embryos resume mitosis significantly earlier than not intact ones regardless of the freezing method (rate: 79% vs. 66%, median time: 7.6 h vs 14.6 h, respectively, p = 0.03). Regarding morphometrics, slow frozen embryos showed lower TCV and higher blastomere symmetry after thawing than vitrified embryos despite having similar blastomere number. IR was related to blastomere number at cryopreservation in slow frozen embryos, but not in vitrified ones.

Conclusions

Interestingly, vitrified/warmed embryos undergo mitosis resumption and compaction significantly earlier than slow frozen/thawed embryos. However, the clinical use of this morphokinetic parameters still remains to be investigated in larger studies.

Trial registration

Retrospectively registered on December 15, 2015 NCT02639715.

     

Endoglucanase enzymatic modification of kraft pulp during recycling

Objective

To investigate the cellulose modification process on kraft pulp during recycling by mono-endoglucanase.

Results

Pichia pastoris expressing endoglucanase, EG1, was grown in a 10 l fermenter yielding a high carboxymethyl cellulase (CMCase) activity of 340 U mg^?1. EG1-mediated modification of kraft pulp resulted in a paper sheet with the tensile index and burst index increased by 10 and 6.5 %, respectively. The kink index (indicating abrupt bends in fibres) of the enzyme-treated group decreased sharply by 45 % after the first recycling, compared with a reduction of only 1 % in the control group. Furthermore, EG1 treatment decreased the growth of crystallinity from 73.5 to 73.2 % and crystal size from 7.45 to 7.21 nm, which alleviated paper aging.

Conclusion

Endoglucanase EG1 modifies the interfacial properties of fibers, which affects fibre morphology during the recycling process and improves the technical properties of the resulting pulp and paper.

     

R-wave synchronised atrial pacing in pediatric patients with postoperative junctional ectopic tachycardia: the atrioventricular interval investigated by computational analysis and clinical evaluation

Background

R-wave synchronised atrial pacing is an effective temporary pacing therapy in infants with postoperative junctional ectopic tachycardia. In the technique currently used, adverse short or long intervals between atrial pacing and ventricular sensing (AP–VS) may be observed during routine clinical practice.

Objectives

The aim of the study was to analyse outcomes of R-wave synchronised atrial pacing and the relationship between maximum tracking rates and AP–VS intervals.

Methods

Calculated AP–VS intervals were compared with those predicted by experienced pediatric cardiologist.

Results

A maximum tracking rate (MTR) set 10 bpm higher than the heart rate (HR) may result in undesirable short AP–VS intervals (minimum 83 ms). A MTR set 20 bpm above the HR is the hemodynamically better choice (minimum 96 ms). Effects of either setting on the AP–VS interval could not be predicted by experienced observers. In our newly proposed technique the AP–VS interval approaches 95 ms for HR > 210 bpm and 130 ms for HR < 130 bpm. The progression is linear and decreases strictly (? 0.4 ms/bpm) between the two extreme levels.

Conclusions

Adjusting the AP–VS interval in the currently used technique is complex and may imply unfavorable pacemaker settings. A new pacemaker design is advisable to allow direct control of the AP–VS interval.

     

Evaluation of cervical posture improvement of children with cerebral palsy after physical therapy based on head movements and serious games

Background

This paper presents the preliminary results of a novel rehabilitation therapy for cervical and trunk control of children with cerebral palsy (CP) based on serious videogames and physical exercise.

Materials

The therapy is based on the use of the ENLAZA Interface, a head mouse based on inertial technology that will be used to control a set of serious videogames with movements of the head.

Methods

Ten users with CP participated in the study. Whereas the control group (n = 5) followed traditional therapies, the experimental group (n = 5) complemented these therapies with a series of ten sessions of gaming with ENLAZA to exercise cervical flexion–extensions, rotations and inclinations in a controlled, engaging environment.

Results

The ten work sessions yielded improvements in head and trunk control that were higher in the experimental group for Visual Analogue Scale, Goal Attainment Scaling and Trunk Control Measurement Scale (TCMS). Significant differences (27% vs. 2% of percentage improvement) were found between the experimental and control groups for TCMS (p < 0.05). The kinematic assessment shows that there were some improvements in the active and the passive range of motion. However, no significant differences were found pre- and post-intervention.

Conclusions

Physical therapy that combines serious games with traditional rehabilitation could allow children with CP to achieve larger function improvements in the trunk and cervical regions. However, given the limited scope of this trial (n = 10) additional studies are needed to corroborate this hypothesis.

     

Cathepsin B inhibitor improves developmental competency and cryo-tolerance of in vitro ovine embryos

Background

Cathepsin B is a lysosomal cysteine protease involved in apoptosis and oocytes which have lower developmental competence show higher expression of Cathepsin B. Furthermore, expression of Cathepsin B show a decreasing trend from oocyte toward blastocyst stage.

Results

Present study assessed the effect of cathepsin B inhibitor, E-64, on developmental competency and cryo-survival of pre-implantation ovine IVF derived embryos. Cathepsin B inhibitor was added during day 3 to 8 of development. One μM E-64 was defined as the optimal concentration required for improving blastocyst rate. This concentration also reduced DNA fragmentation and BAX as apoptotic markers while increasing total cell number per blastocyst and improving anti-apoptotic marker, the BCL2. We further showed that addition of 1.0 μM of E-64 during day 3 to 8 of development improved re-expansion and hatching rates of blastocysts post vitrification. E-64 also reduced rate of DNA fragmentation and BAX expression and increased total cell number per blastocyst and BCL2 expression post vitrification. However, addition of E-64 post vitrification reduced the hatching rate.

Conclusion

Therefore, it can be concluded that inhibition of cathepsin B in IVC, not only improves quality and quantity of blastocysts but also improves the cryo-survival of in vitro derived blastocysts.

     

Dexamethasone downregulates SIRT1 and IL6 and upregulates EDN1 genes in stem cells derived from gingivae via the AGE/RAGE pathway

Objectives

To evaluate the effects of dexamethasone on the aging of mesenchymal stem cells from human gingiva using next-generation sequencing.

Results

Four mRNAs were upregulated and 12 were downregulated when the results of dexamethasone at 24 h were compared with the control at 24 h. Expressions of SIRT1 and IL6 were decreased in dexamethasone at 24 h but expression of EDN1 was increased.

Conclusions

Application of dexamethasone reduced the expression of SIRT1 and IL6 but enhanced the expression of EDN1 of stem cells.

     

Safety and efficacy of transseptal puncture guided by real-time fusion of echocardiography and fluoroscopy

Aims

Visual guidance through echocardiography and fluoroscopy is crucial for a successful transseptal puncture (TSP) in a prespecified region of the fossa ovalis. The novel EchoNavigator system Release II (EchoNav II, Philips Healthcare, Andover, Massachusetts, USA) enables the real-time fusion of fluoroscopic and echocardiographic images. We evaluated this new imaging method in respect to safety and efficacy of TSP during MitraClip implantation and left atrial appendage closure.

Methods

Forty-four patients before (?EchoNav) and 44 patients after (+EchoNav) the introduction of real-time fusion were included in our retrospective, single-centre study. The primary endpoint was the occurrence of adverse events due to TSP. Secondary endpoints were successful puncture at the prespecified region and time until TSP (min).

Results

In both groups TSP was performed successfully in the prespecified region and no adverse events occurred during or due to the accomplishment of TSP. Time until TSP was significantly reduced in the +EchoNav group in comparison with the EchoNav group (18.48 ± 5.62?min vs. 23.20 ± 9.61?min, p = 0.006).

Conclusions

Real-time fusion of echocardiography and fluoroscopy proved to be as safe and successful as standard best practice for TSP. Moreover, efficacy was improved through significant reduction of time until TSP.

     

Dichorionic triplets following frozen-thawed poor-stage embryo transfer: a report of two cases and a review

Background

We describe two cases of dichorionic triplet pregnancy after a frozen-thawed poor-stage embryo transfer.

Main body of the abstract

A 39-year-old and a 41-year-old woman underwent ART treatment. The first patient underwent intracytoplasmic sperm injection (ICSI) at 34 years of age, and two frozen-thawed poor-stage embryos were transferred at 39 years of age with assisted hatching, resulting in a trichorionic triamniotic triplet pregnancy. The second patient underwent ICSI, and two poor-grade blastocysts were transferred followed by assisted hatching, resulting in a dichorionic triamniotic triplet pregnancy.In the first case, the heartbeat of one monozygotic twin fetus had stopped on day 48 post-transfer (9 weeks 2 days), resulting in a dichorionic diamniotic twin pregnancy. A healthy boy and girl were delivered by elective caesarean section at 36 weeks, 5-days gestation. In the second case, the patient underwent selective reduction of the monochorionic twins, resulting in a single pregnancy that was vaginally delivered without any problems at 38 weeks 0-days gestation.

Short conclusions

Numerous factors may be associated with the development of a monochorionic pregnancy; however, controversies still remain. The present morphological grading for embryos is insufficient for inhibiting the development of a monochorionic pregnancy.