extfieldsofstudy: Medicine Biology

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C. elegans germline cell death, live!

Ironically, oogenesis, the developmental process that generates the female gamete, the major conveyor of germline immortality across generations, involves a fateful decision—to commit suicide or to complete development. In organisms as diverse as worms, flies, and mice, oocyte precursors, oogonia, are generated in excess of the number of fully grown oocytes produced for fertilization. This surfeit is culled by apoptosis, also called programmed cell death [1–3], which is mediated by the core apoptotic machinery comprised of CED-9/Bcl2, CED-4/Apaf-1, and the CED-3/Caspase [4]. Apoptosis in the female germline has been proposed to play two major functions: a nurse cell function, in which the oogonia chosen for apoptosis provide their cytoplasm and organelles to other developing oocytes, and a quality control function, in which cells containing a damaged genome or dysfunctional organelles (especially mitochondria) are culled. In Caenorhabditis elegans, more than half of all female germ cells undergo apoptosis [1,5]. A key unanswered question in the field is how female germ cells are chosen to adopt the apoptotic fate in the correct numbers. This issue has been challenging to address because the characteristic refractile appearance of apoptotic germ cells corresponds to a late stage in the process occurring during or after the engulfment of the dying germ cell by neighboring somatic gonadal cells, the sheath cells (Fig 1A). The paper by Raiders and colleagues in this issue of PLOS Genetics reports a tour-de-force analysis of early events in the cell biology of germline apoptosis using multiple microscopic modalities, including live-cell imaging conducted in the wild-type and engulfment-defective mutants [6]. The breathtaking images and videos give us our earliest views into the execution of the apoptotic program in female germ cells and provide strong evidence that apoptosis serves both a nurse cell function and a quality control mechanism. Raiders and colleagues used a combination of live imaging and an examination of fixed samples by fluorescence and transmission electron microscopy (TEM) to elucidate cellular events occurring during the execution stage of the apoptotic program. They noticed that dying germ cells were devoid of mitochondria. Using live-cell imaging, they observed the selective export of mitochondria from doomed germ cells into the cytoplasmic core of the germline (Fig 1B). Mitochondrial export is dependent on CED-3/Caspase activity and represents the earliest described cellular event in germline apoptosis. Mitochondrial export appears to occur along radial microtubules extending through the ring channels and requires the plus-end–directed motor kinesin, consisting of the UNC-119 kinesin heavy chain and the KLC-1 kinesin light chain. The authors show that the mitochondrial Rho GTPase (Miro), which is involved in

Ironically, oogenesis, the developmental process that generates the female gamete, the major conveyor of germline immortality across generations, involves a fateful decision-to commit suicide or to complete development. In organisms as diverse as worms, flies, and mice, oocyte precursors, oogonia, are generated in excess of the number of fully grown oocytes produced for fertilization. This surfeit is culled by apoptosis, also called programmed cell death [1][2][3], which is mediated by the core apoptotic machinery comprised of CED-9/Bcl2, CED-4/Apaf-1, and the CED-3/Caspase [4]. Apoptosis in the female germline has been proposed to play two major functions: a nurse cell function, in which the oogonia chosen for apoptosis provide their cytoplasm and organelles to other developing oocytes, and a quality control function, in which cells containing a damaged genome or dysfunctional organelles (especially mitochondria) are culled.
In Caenorhabditis elegans, more than half of all female germ cells undergo apoptosis [1,5]. A key unanswered question in the field is how female germ cells are chosen to adopt the apoptotic fate in the correct numbers. This issue has been challenging to address because the characteristic refractile appearance of apoptotic germ cells corresponds to a late stage in the process occurring during or after the engulfment of the dying germ cell by neighboring somatic gonadal cells, the sheath cells ( Fig 1A). The paper by Raiders and colleagues in this issue of PLOS Genetics reports a tour-de-force analysis of early events in the cell biology of germline apoptosis using multiple microscopic modalities, including live-cell imaging conducted in the wild-type and engulfment-defective mutants [6]. The breathtaking images and videos give us our earliest views into the execution of the apoptotic program in female germ cells and provide strong evidence that apoptosis serves both a nurse cell function and a quality control mechanism.
Raiders and colleagues used a combination of live imaging and an examination of fixed samples by fluorescence and transmission electron microscopy (TEM) to elucidate cellular events occurring during the execution stage of the apoptotic program. They noticed that dying germ cells were devoid of mitochondria. Using live-cell imaging, they observed the selective export of mitochondria from doomed germ cells into the cytoplasmic core of the germline ( Fig  1B). Mitochondrial export is dependent on CED-3/Caspase activity and represents the earliest described cellular event in germline apoptosis. Mitochondrial export appears to occur along radial microtubules extending through the ring channels and requires the plus-end-directed motor kinesin, consisting of the UNC-119 kinesin heavy chain and the KLC-1 kinesin light chain. The authors show that the mitochondrial Rho GTPase (Miro), which is involved in mitochondrial transport in neurons [7], is not required for selective mitochondrial export. Thus, this mitochondrial export occurs by a new mechanism. Mitochondria are maternally inherited in all but a few animal species. Evidence suggests that a process of purifying selection operates during oogenesis to ensure the inheritance of maximally fit mitochondria and to protect against mitochondrial bottlenecks during germline development [8]. Interestingly, Raiders and colleagues noticed that, in a few instances, apoptotic germ cells contained one mitochondrion or a few clumped mitochondria. It is tempting to speculate that selective mitochondrial export functions as part of the purifying selection process by recycling the most active mitochondria into the germline pool but relegating inactive or damaged mitochondria to the dust heap with the dying germ cell corpse. Further work will be needed to test this hypothesis and unravel the molecular mechanisms of selective mitochondrial export. During apoptotic execution, Raiders and colleagues observed two stages of cell shrinkage. The first, referred to as "fast shrinkage," commences approximately 80 min before the dying germ cell exhibits optical refractility. By contrast, "slow shrinkage" occurs following the closure of the ring channels (Fig 1B). During fast shrinkage, PGL-1, a defining component of perinuclear ribonucleoprotein particles called "P granules," which are associated with nuclear pore complexes and represent the primary sites of mRNA export to support oogenesis [9], disappears from the undone germ cells. Despite the loss of PGL-1, P granules were observed by TEM until the final stages of apoptosis. Whether the loss of PGL-1 and perhaps associated P granule components reflects their trafficking back to the cytoplasmic core for reuse remains to be determined. Nonetheless, the slow shrinkage and the selective export of mitochondria likely represent the final gasp of the dying cell in fulfilling its role as a nurse cell, which is terminated upon the closure of the ring channels (Fig 1B).
Upon closure of the ring channels, the dying cell extensively remodels its cytoskeleton. Microtubule "cocoons," closely apposed to the cell cortex and the nucleus, form rings in the apoptotic cells soon after ring channel closure. The apoptotic cells also extensively reorganize their actin cytoskeletons by forming large cofilin-actin rods approximately 40-70 min after ring channel closure. Interestingly, cofilin-actin rods have been observed in several neurodegenerative diseases including Alzheimers and Huntingtons disease. Thus, not only does the dying cell actively participate in its own demise, but it erects cytoskeletal elements that are defining features of the process. These cytoskeletal changes may reinforce the structure of the dying cell to protect the germline or the engulfing cell from degradative activities, including activated caspases.
An unexpected finding of the study is that binucleate and trinucleate germ cells appear to form as byproducts of morphogenesis of the syncytium. These aberrant multinucleate germ cells are eliminated by apoptosis. This observation documents a clear role for germline apoptosis in serving a quality control mechanism. The mechanisms that surveil and recognize multinucleate germ cells and trigger CED-3 caspase activation remain to be determined.
The work of Raiders and colleagues illustrates the continued power of descriptive studies in developmental biology. New microfluidic methods may enable longer term observation of the apoptotic process [10]. These invaluable insights and tools will help researchers solve the mystery of how germ cells make their fateful choice between life and death.


extfieldsofstudy: Medicine Chemistry

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Codon-optimized DsRed fluorescent protein for use in Mycobacterium tuberculosis

Objective We have previously codon-optimized a number of red fluorescent proteins for use in Mycobacterium tuberculosis (mCherry, tdTomato, Turbo-635). We aimed to expand this repertoire to include DsRed, another widely used and flexible red fluorescent protein. Results We generated expression constructs with a full length DsRed under the control of one of three strong, constitutive promoters (Phsp60, PrpsA or PG13) for use in mycobacteria. We confirmed that full length DsRed (225 amino acids) was expressed and fluoresced brightly. In contrast to mCherry, truncated versions of DsRed lacking several amino acids at the N-terminus were not functional. Thus, we have expanded the repertoire of optimized fluorescent proteins for mycobacteria.

Introduction
Fluorescent proteins (FPs) have become the work horses of molecular biology and microbiology, with numerous applications. A plethora of variants of Aequorea victoria green fluorescent protein (GFP) [1] and Discosoma sp red fluorescent protein (DsRed) [2] are available covering almost the whole light spectrum from green to infrared [3]. Mutant derivatives have been engineered with altered excitation and emission wavelengths, increased or decreased stability, resistance to photo bleaching, sensitivity to environmental stimuli and substrates, as well as time for fluorophore maturation, intrinsic brightness and multimeric formats [3,4]. We previously described the use of a range of red reporters, of which the brightest was mCherry [5]. We wanted to expand our repertoire of proteins. Since DsRed has been widely used as a bright and stable reporter, we optimized constructs for its expression in M. tuberculosis.

BMC Research Notes
*Correspondence: Tanya.Parish@idri.org 2 Infectious Disease Research Institute, Seattle, WA 98102, USA Full list of author information is available at the end of the article GAG TTC together with the reverse primer DsRed-R 5′-AAG CTT TTA CAG GAA CAG GTG GTG CCG-3′. The restriction sites are underlined, potential start codons are in bold. The ORFs were excised and cloned into pSMT3 [6] as BamHI/HindIII fragments to generate pBlazeA1, pBlazeB1 and pBlazeC1 with DsRed under the control of the hsp60 promoter (Table 1). Plasmids pBlazeC8 and pBlazeC10 were generated by replacing P hsp60 with P rpsA and P G13 respectively. All three promoters should drive constitutive high level expression [5,7,8].

Quantitation of fluorescence in whole cells
Mycobacterium tuberculosis was electroporated as described [9] and transformants selected with hygromycin. M. tuberculosis was grown to stationary phase, harvested, washed twice in 10 mM Tris pH 8.0 and resuspended in 10 mM Tris pH 8.0 to an OD 580 of 0.25, 0.10, 0.05 and 0.01 in 12 × 100 mm glass culture tubes. Fluorescence was measured on a Shimadzu RF-1501 spectrofluorimeter (Shimadzu) with a detection range of 0-1015 relative fluorescent units at Ex/ Em 558/583 nm [5].

Western analysis of fluorescent proteins
Cell extracts were prepared from liquid cultures. Cells were harvested by centrifugation, washed twice in 10 mM Tris (pH 8.0), resuspended in 1 ml of 10 mM Tris (pH 8.0), and added to lysing matrix B tubes (QBiogene). Cells were disrupted using the Fastprep (QBiogene) set at speed 6.0 for 30 s. Samples were centrifuged at 4000 rpm for two min, and the supernatant was recovered and filter sterilized (0.2 micron filter). Protein was quantified using a BCA kit (Pierce), and 10 μg of total protein was subjected to Western blot using a rabbit anti-body (Clonetech). The primary antibody was detected using horseradish peroxidase goat-anti-rabbit (Sigma), and activity was detected using an ECL kit (GE Healthcare).

Results
We were interested in the use of FPs in M. tuberculosis and had previously used these as reporters of bacterial viability for in vitro and in vivo studies [5,8]. We were successful in obtaining high level expression by using codon-optimized versions of red fluorescent proteins driven by strong mycobacterial promoters [5].

Optimization of DsRed expression
We wanted to expand the range of reporters available for use to increase flexibility and allow dual reporter expression and monitoring. We selected DsRed for optimization, based on its Ex/Em wavelengths, and the fact that it is a well-characterized FP in wide use [3,4,[10][11][12][13][14].

Expression of DsRed uses a different translational start site than mCherry
Our initial attempts to obtain expression of a codonoptimized DsRed were unsuccessful. We constructed a synthetic gene for DsRed using a similar approach as we used with another red fluorescent protein mCherry (Fig. 1). We designed a codon-optimized version based on the DsRed-T3 protein previously used. We cloned the synthetic version into a mycobacterial expression vector and tested for fluorescence in M. tuberculosis. Surprisingly, we did not detect any fluorescence from this construct (Fig. 1c).
mCherry is a variant of DsRed and we expected the two proteins would be similarly functional. Our previous work demonstrated that mCherry is expressed from a distal translational start site than the one annotated in the databases [15]. Sequence alignment shows the few mutations which differ between the two (Fig. 2a); these include loss of the translational start site we identified for mCherry, although there are still multiple translational start sites (Fig. 1a). The version we used for the  synthetic gene used a downstream translation start site and would produce a truncated version of DsRed as compared to mCherry. Therefore it was possible that we did not express the full protein (Fig. 1b). In order to determine the functional start site for DsRed we used a different approach in which we cloned several versions of the  [19]. b Activity of full length DsRed expressed from mycobacterial promoters. pBlazeC1-P hsp60 ; pBlazeC8-P rpsA ; pBlazeC10-P G13 . Recombinant M. tuberculosis was resuspended in 10 mM Tris pH 8.0 to an OD 580 of 0.25, 0.10, 0.05 and 0.01 in 12 × 100 mm glass culture tubes. Fluorescence was measured at Ex/Em 558/583 nm. Data are the average ± SD of three cultures. c Plasmids were transformed into E. coli and cell-free extracts analyzed by Western blotting; 10 µg protein were subjected to SDS-PAGE, blotted onto PVDF membrane and visualized with anti-DsRed antibody. Lane 1-E. coli (no plasmid); Lane 2-pRed1; Lane 3-pRedA1; Lane 4-pRedB1; Lane 5-pRedC1; Lanes 6 and 7-pBlazeC1; Lane 8-pBlazeC8; Lane 9-pBlazeC10. The arrow indicates the size of the DsRed protein coding region into the expression vector under the control of the constitutive hsp60 promoter (Fig. 2b).
In order to test this, we used PCR amplification to extend the region sequentially. We extended the gene to incorporate both additional start sites and generate proteins of 214, 220 and 225 amino acids. These variants were cloned into the same mycobacterial expression system and tested. Plasmids were transformed into M. tuberculosis and fluorescence was monitored. In contrast to mCherry, expression of a functional fluorescent DsRed was not seen with any truncated versions of the protein.
In fact fluorescence could only be detected when the full length amino acid sequence (as annotated) was cloned into the expression vector; high level fluorescence was seen with transformants carrying the plasmid pBlazeC1 (Fig. 2b).
We constructed two alternative vectors with DsRed under the control of either P rpsA or P G13 (pBlazeC8 and pBlazeC10 respectively); both of these constructs gave high level expression in M. tuberculosis. Western blotting using an anti-DsRed antibody in E. coli demonstrated that a protein of the expected size was only seen in bacteria carrying the full length construct (pBlazeC series), but not in the strains carrying the truncated version ( Fig. 2c; lanes 5-9). Two additional bands are see in the Western, these are unknown proteins, but are also present in the control E. coli (no plasmid, Fig. 2c, lane 1).

Discussion
We have determined that the functional translational start sites for two closely related FPs are different in M. tuberculosis. Although mCherry was functional even when a truncated version was being expressed, DsRed was non-functional in a truncated form and only fluoresced when expressed as a full length protein (225 amino acids). Western blotting suggested that the lack of fluorescence was most likely due to a lack of protein expression, since proteins could not be detected in the plasmids carrying truncated forms. This difference may relate to protein stability, with the extended N-terminal portion of DsRed increasing stability or protein maturation; alternatively this could be attributed to the physical state of the active proteins, since mCherry functions as a monomer, whereas DsRed is a tetramer which might also affect protein degradation.
Fluorescent proteins have proved useful in multiple applications in mycobacteria; our previous constructs using mCherry have been widely disseminated to the community and used in a range of methods. For example, we have used these for high throughput drug testing [16], and imaging infection using animal models [8]. Other approaches have used mCherry to develop reporter strains for environmental sensing [17].
In conclusion, we have codon-optimized DsRed for use in M. tuberculosis and demonstrated its high level fluorescence in that species from three different promoters of slightly varying strength (hsp60, rpsA, and G13). These vectors extend our current repertoire of functional fluorescent proteins for mycobacteria. They will be useful for generating fluorescent strains of M. tuberculosis for use in multiple studies, such as monitoring drug efficacy in vitro and in vivo [5,8,16,18] and will allow for detection of multiple reporters simultaneously.


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The complete chloroplast genome sequence of the plant Euonymus fortunei (Celastraceae)

Abstract Chloroplast (cp) genome sequences become a useful popular tool for population and phylogeny in recent reports. Here, the complete cp genome of the Euonymus fortunei has been reconstructed from the whole-genome Illumina sequencing data. The circular genome is 157,639 bp in size, and comprises a pair of inverted repeat (IR) regions of 26,668 bp each, a large single copy (LSC) region of 85,956 bp and a small single copy (SSC) region of 18,347 bp. The total GC content is 37.2%, while the corresponding values of the LSC, SSC, and IR region are 35.1%, 31.7%, and 42.7%, respectively. The cp genome contains 126 genes, including 89 protein-coding genes, eight ribosomal RNA genes, and 29 transfer RNA genes and one pseudogene. The maximum-likelihood phylogenetic analysis showed a strong sister relationship with Euonymus japonicus in Celastraceae. Our findings provide a foundation for further investigation of cp genome evolution in E. fortunei and other higher plants.

The chloroplast (cp) contains its autonomously replicating DNA genome that proceeds with photosynthesis and many other biological activities such as synthesizing starch, fatty acids, and other proteins relative to its special functions (Ohyama et al. 1986;Bausher et al. 2006). Most cp genomes are circular DNA molecules which have the typical quadripartite structure containing two repeat regions (IRa and IRb), LSC, and SSC (Yang et al. 2010).
Euonymus fortunei (Celastraceae), an evergreen shrub, is native to East Asia including China, Korea, and Japan. As an ornamental plant, E. fortunei has a wide range of applications in vertical garden and soil and water conservation. In addition, E. fortunei also serves as a traditional medicine for the treatment of inflammation and neurological diseases (Jian 2007;Zuo et al. 2012). To facilitate its genetic studies and thus contribute to its development and sustainable utilization, we assembled its cp genome using high-throughput Illumina sequencing technology in this study, as well as analysed its phylogenetic evolution, which will be helpful for better understanding of evolution within the Celastraceae and further studies on its molecular breeding and genetic engineering.
DNA extraction form the fresh leaves was collected from a single individual of E. fortunei in Xi'an Botanical Garden (Xi'an, Shaanxi Province, China). High-throughput DNA sequencing was conducted on the Illumina HiSeq 2500 Sequencing System (Illumina, San Diego, CA) by Breeding Biotechnologies (Breeding, Yangling, China). Total 16.8 M 150 bp raw reads were retrieved and trimmed by CLC Genomics Workbench v8.0 (CLC Bio, Aarhus, Denmark). A subset of 12.6 M trimmed reads were used for reconstructing the cp genome by NOVOPlasty (Dierckxsens et al. 2016), with that of its congener Euonymus schensianus (GenBank: NC_036019.1) as the initial reference genome. A total of 23,125,435 individual cp reads yielded an average coverage of 594.8-fold. The cp genome was annotated in GENEIOUS R9 (Biomatters Ltd., Auckland, New Zealand) by aligning with that of E. schensianus (NC_036019.1) and was drawn to the circular cp genome sequence map of OGDRAW 1.1.
The cp genome of E. fortunei is a double-stranded circular DNA molecule with 157,639 bp in size (MH150885). It comprises a pair of inverted repeat (IR) regions of 26,668 bp each, separated by a large single copy (LSC) region of 85,956 bp and a small single copy (SSC) region of 18,347 bp. The total GC content is 37.2%, while the corresponding values of the LSC, SSC, and IR region are 35.1%, 31.7%, and 42.7%, respectively.
This cp genome harbours 126 functional genes, including 89 protein-coding genes (PCGs), 29 tRNA genes, and eight rRNA genes. Eighteen PCGs, eight tRNA genes, and all rRNA genes are duplicated in the IR regions. The LSC region possesses 59 PCGs and 20 tRNA genes, while the SSC region contains 12 PCGs and one tRNA gene. Thirty three PCGs, 14 tRNA genes, and four rRNA genes are located in the forward   strand while others are located in the reverse strand. Moreover, 14 genes contain one intron, while ycf3 harbours two introns; all the other genes are intronless. Among those genes, 44 are involved in photosynthesis, and 53 genes are involved in self replication. This is similar to those previously reported for the cp genomes of most other vascular plants (Yang et al. 2014).
Sixty PCGs data among 41 cp sequences were aligned by MAFFT (Katoh et al. 2002) and then were connected as gene strings. The maximum-likelihood phylogenetic tree of E. fortunei was generated using those gene strings sequence by MEGA 6.0 (Tamura et al. 2013) with using 500 bootstrap replicates (Figure 1). The phylogenetic analysis showed the position of E. fortunei was situated as the sister of E. japonicus and E. schensianus in Celastraceae. Our findings provide a foundation for further investigation of cp genome evolution in E. fortunei and other higher plants.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Fundamental Research Funds from Shaanxi Province of Education [17JK0182].


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CpG-set association assessment of lipid concentration changes and DNA methylation

Epigenome association studies that test a large number of methylation sites suffer from stringent multiple-testing corrections. This study’s goals were to investigate region-based associations between DNA methylation sites and lipid-level changes in response to the treatment with fenofibrate in the GAW20 data and to investigate whether improvements in power could be obtained by taking into account correlations between DNA methylation at neighboring cytosine-phosphate-guanine (CpG) sites. To this end, we applied both a recently developed block-based data-dimension-reduction approach and a region-based variance-component (VC) linear mixed model to GAW20 data. We compared analyses of unrelated individuals with familial data. The region-based VC approach using unrelated (independent) individuals identified the gene LGALS9C as significantly associated with changes in triglycerides. However, univariate tests of individual CpG sites yielded no valid statistically significant results.

Background
Lipid levels can be influenced by drug therapy or lifestyle factors such as diet, physical activity, alcohol consumption, and smoking [1]. Lipid levels are also associated with inherited genetic variants (single-nucleotide polymorphisms [SNPs]), as revealed by several genome-wide association studies [2]. However, DNA sequence variation explains only a small proportion of lipid-level variance [2]. Epigenetic modifications (eg, DNA methylation) alter DNA accessibility and hence can be involved in regulating patterns of gene expression. Through regulation of lipid levels, epigenetic mechanisms may contribute to cardiovascular risk profiles [3,4]. Irvin et al. [3] identified strong association of 4 cytosine-phosphate-guanine (CpG) sites within the CPT1A gene on chromosome 11 with both triglycerides (TGs) and very-low-density lipoprotein C (VLDL-C). Because their analysis examined phenotype associations at each CpG site, a substantial correction for multiple testing was required.
This study's goals were to investigate region-based associations between DNA methylation sites and lipid-level changes in response to the treatment with fenofibrate in the real data set provided by the GAW20 and to investigate whether improvements in power could be obtained by taking into account correlation between DNA methylation at neighboring CpG sites. We conducted 2 complementary block-based association tests that simultaneously accommodated all CpG sites that fell within a genomic region (block) for the purpose of investigating whether taking into account the correlation between DNA methylation at neighboring CpG sites and reducing the number of tests can improve power.
Turgeon et al. [5] have proposed "principal components of explained variation (PCEV)." The PCEV approach integrates, simultaneously, an optimal data-dimension-reduction technique with testing for association. It provides analytical and empirical p value calculations for testing association between a set of correlated variables (eg, methylation profiles of a genomic region) and 1or more variables of interest (eg, high-density lipoprotein [HDL]/ TG).
We contrast PCEV with a variance components (VC) score test method. This is a sequence kernel association test (SKAT)-type association test that decomposes the total variance of a phenotype (eg, HDL/TG) into the variance explained by a block/region-methylation profiles and a residual variance term [6]. Specifically, the model assumes that the phenotypic similarity between subjects is captured by the region-methylation similarity. The VC-score approach significantly reduces the model degrees of freedom compared to standard multivariate regression models.

Methods
Suppose we have observed data {Y, x, C} where Y is an n × p matrix of n subjects for which a block of p variables/phenotypes are measured (eg, methylation values at a genomic region/gene with pCpG dinucleotides), x is an n × 1 vector of an observed trait of interest (eg, HDL phenotype) and C is an n × r matrix where the columns are known confounding factors (eg, age, sex).

PCEV approach
PCEV is a dimension-reduction technique that searches for a linear combination (a principal component) of the columns of Y, y pcev = Y w (w is ap × 1 vector), that maximizes h 2 (w), the ratio of the variance in Y explained by x to the total variance of Y, while taking into account the confounding factors, C. This new score y pcev can then be used as a phenotype in standard statistical models to test for the relationship between Y and x. Searching for y pcev is equivalent to projecting the rows of Y into w, where w is the most relevant direction in p-dimension space to x. A linear relationship between Y and x can be tested by H 01 : corr(y pcev , x) = 0. This test requires the use of the data twice, and therefore a naïve approach for p value calculation will suffer from Type I error inflation. However, the null H 01 is equivalent to testing for H 02 : h 2 (w pcev ) = 0 which uses the data only once. Turgeon et al. [5] derived an analytic test for the null hypothesis H 02 , which was shown to yield the proper Type I error rate.

VC-score approach
In a reverse model where x (eg, HDL) is modeled as the response variable and Y as a design matrix of p predictors, the VC model links x to Y using a linear mixed-effects regression model in which Y has an effect on the variance of x instead of on its mean [6]. This approach was developed to test association between a set of rare variants and a phenotype of interest. However, the test can be adapted easily to handle different types of design matrices, such as methylation from a genomic region of interest. This method can be extended to take into account population and family structures. The family-based VC-score approach is a linear mixed-effects model in which a second random effect for genetic relationships (ie, kinship) is added [7].
Phenotypic, methylation, and covariate data Circulating blood lipids, HDL, TGs, and the methylation profiles were measured at baseline and following 3 weeks of daily treatment with 160 mg of micronized fenofibrate [2]. For this study, we investigated HDL and TG changes among 714 participants for whom pretreatment methylation data were available. Because the PCEV approach has only been implemented for use with independent subjects, analyses using this method were conducted using 242 unrelated individuals. The selection of the maximum set of unrelated individuals from each pedigree was done using a greedy algorithm that used the kinship matrix to sequentially remove related individuals [8]. Log-transformations were performed for TG, as this variable was not normally distributed.
T-cell pre-and posttreatment DNA-methylation at 463,995CpG sites were already normalized using ComBat [3]. These CpG sites were allocated to 22,319 genes. We also included sites located 20 kb up-and downstream of the gene region. Only the CpG sites with gene annotations were evaluated in the analyses; consequently, we analyzed 401,326 CpG sites. Because PCEV works when the block and sample sizes are comparable [5], we divided the largest gene blocks to obtain 22,488 gene regions with no more than 130 CpG sites per block.
We focused on the pretreatment methylation levels to evaluate the effect of individual CpG sites and genes on explaining the observed heterogeneity in response to treatment. To capture unwanted variability in methylation profiles, which could result from variation in cell purity or batch effects, we constructed principal components of genome-wide methylation levels using 2000 randomly sampled probes from all autosomes. The association analyses between pretreatment methylation probes and blood lipid changes were adjusted for age, sex, study center, smoking status, diagnosed metabolic syndrome status, the fast time on the pre-and posttreatment visits, and the top 4 methylation-derived principal components (PCs).

Results
Bonferroni thresholds for significance at a 10% family-wise error rate were established using 401,326 univariate tests and 22,488 CpGset tests. No univariate tests for TG changes passed this threshold. The family-based VC-score approach identified 2 genes (RNMT andMIR130B) as significantly associated with HDL changes. The VC-score approach using unrelated subjects identified the gene LGALS9C as significantly associated with TG changes. Table 1 lists the 5 most significant genes with association to lipid changes identified by univariate, PCEV and VC-score approaches. Among the top 5 genes, both region-based approaches identified NUDCD3 for its relationship with HDL changes among independent participants. There is no overlap among the top 5 genes for TG changes identified by the 3 approaches. Figure 1 shows quantile-quantile (Q-Q) plots for the gene-based p values and the individual CpG-based p values for the HDL changes and TG changes, using data from the 242 unrelated individuals. Under each analysis, adjustments with and without the 4 methylation PCs were compared, revealing that inclusion of these PCs was important in controlling for unknown confounding. Without this extra adjustment, the distribution of p values was very biased away from what would be expected under the null. Figure 2 contrasts the results obtained using the independent participants and the ones

Discussion
In this study, we investigated associations between DNA methylation and lipid-level changes in response to fenofibrate treatment in the GAW20 real data set. The smallest p value found by both the VC-score test for TG differences and the single-CpG association test was at gene LGALS9C. However, as a result of multiple-testing power-loss issues, no single-CpG test passed a Bonferroni-corrected threshold. Simulation studies are necessary for more rigorous power assessments comparing region-based and univariate methods. Furthermore, we did not replicate the results of Irvin et al. [3] for baseline lipids, although this is not surprising as we analyzed lipid-level changes. We focused on linear relationships; however, nonlinear association methods may be more favorable/powerful when the primary interest is lipid-level changes.
In all analyses, we adjusted for available/known confounders and for unknown confounders using 4 PCs calculated based on 2000 CpG probes selected randomly from available DNA methylation on all chromosomes. We also contrasted this with an analysis using PCs calculated from all CpG sites, and found little difference in the results (not shown). The adjustment resulting from the PCs improved the validity of VC-score test results; however, (unusual) deviation of the PCEV test statistic from the null distribution was also indicated. This might be a consequence of the nonrobustness of PCEV to violation of normality assumption or the constant variance assumption of model residuals (errors). Even after normalization, methylation proportions have variances that are small when means are near 0 and 1. This heteroscedasticity might lead to a loss of power [9]. Thus, a transformation such as the logit may help in obtaining test statistics with valid null distributions.
We considered 2 ways to accommodate the relatedness among the participants: to restrict the analysis to unrelated individuals or to use a linear mixed model that takes family structure into account. In contrast to the well-behaved p value distribution for the analysis of unrelated subjects, the family-based VC-score test showed inflation in the Q-Q plots. Hence, the significant results for the 2 genes RNMT and MIR130B may be questionable. Almeida et al. [10] found that heritability of pretreatment DNA methylation was much higher than expected. Our results agree that pedigree-based kinship corrections are insufficient to correct for familial correlations in DNA methylation, and that additional corrections must be considered.
Other region-based association methods may be worth exploring in the future, such as the global analysis of methylation profiles (GAMP) [11] in which the density of methylation values in a region is approximated by B-splines and then the spline coefficients for each individual are used as covariates in association tests. Other strategies to accommodate the family structure in the region-based association tests include MF-KM (multivariate family data using kernel machine regression) [12], a linear mixed model built upon kernel machine regression, and mFARVAT (multivariate family-based rare variant association tool) [13], a quasi-likelihood-basedscore test approach.

Conclusions
The region-based VC approach using unrelated individuals identified the gene LGALS9C as significantly associated with changes in triglycerides. However, univariate tests of individual CpG sites yielded no valid statistically significant results. After correctly accounting for the unknown confounding and subject relatedness, region-based methods show an improvement in power to detect associated genes as compared to single-marker methods.

Funding
Publication of this article was supported by NIH R01 GM031575. This work was also supported by the Ludmer Centre for Neuroinformatics and Mental Health, the Canadian Institutes of Health Research grant # 130344, the Fonds de recherche du Québec -Santé grant # 31110, and CANSSI, the Canadian Statistical Sciences Institute.

Availability of data and materials
The data that support the findings of this study are available from the Genetic Analysis Workshop (GAW), but restrictions apply to the availability of these data, which were used under license for the current study. Qualified researchers may request these data directly from GAW.

About this supplement
This article has been published as part of BMC Proceedings Volume 12 Supplement 9, 2018: Genetic Analysis Workshop 20: envisioning the future of statistical genetics by exploring methods for epigenetic and pharmacogenomic data. The full contents of the supplement are available online at https://bmcproc.biomedcentral.com/articles/supplements/volume-12-supplement-9.


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From Implantation to Birth: Insight into Molecular Melatonin Functions

Melatonin is a lipophilic hormone synthesized and secreted mainly in the pineal gland, acting as a neuroendocrine transducer of photoperiodic information during the night. In addition to this activity, melatonin has shown an antioxidant function and a key role as regulator of physiological processes related to human reproduction. Melatonin is involved in the normal outcome of pregnancy, beginning with the oocyte quality, continuing with embryo implantation, and finishing with fetal development and parturition. Melatonin has been shown to act directly on several reproductive events, including folliculogenesis, oocyte maturation, and corpus luteum (CL) formation. The molecular mechanism of action has been investigated through several studies which provide solid evidence on the connections between maternal melatonin secretion and embryonic and fetal development. Melatonin administration, reducing oxidative stress and directly acting on its membrane receptors, melatonin thyroid hormone receptors (MT1 and MT2), displays effects on the earliest phases of pregnancy and during the whole gestational period. In addition, considering the reported positive effects on the outcomes of compromised pregnancies, melatonin supplementation should be considered as an important tool for supporting fetal development, opening new opportunities for the management of several reproductive and gestational pathologies.

Introduction
Melatonin is a lipophilic hormone synthesized and secreted mainly in the pineal gland, acting as a neuroendocrine transducer of circadian and circannual photoperiodic [1]. It is released cyclically during over a 24-h period, with a peak during the night [2]. Moreover, melatonin is a potent antioxidant, with greater efficacy than vitamin C and E [3]. In fact, together with its metabolites, this hormone directly removes reactive oxygen species (ROS) [4], increasing the gene expression of several antioxidant enzymes, like superoxide dismutase (SOD) and glutathione (GSH), inhibiting pro-oxidative enzymes, and reducing cellular oxidative damage. Melatonin is synthesized in high concentrations in the mitochondria, constituting one of the most important factors for ROS elimination [5].
In addition to having antioxidant function, melatonin plays a key role in several physiological functions, such as in the circadian rhythm [6].
The circadian rhythm is present in almost all the organisms, and organizes the physiological systems in order of time, aligning them with the 24-h environmental cycles. In fact, it is involved in a series of physiological processes, such as sleep/wake [7], physique temperature regulation [8], and hormone secretion [9]. In mammals, circadian rhythm regulation is based on a series of feedbacks between groups of genes known as "clock genes", expressed in almost all tissue types [10]. Within the circadian rhythm, melatonin participates through the regulation of critical gene expressions [11].
Melatonin is also synthetized in other tissues, such as placental tissue, retina, brain, bone marrow, and lymphocytes, thereby regulating local physiological processes [12].
Several studies have displayed how the interruption of the circadian rhythm through night-time work, which causes exposure alteration to light, negatively affects the implantation and pregnancy success at a molecular level, triggering an increase in infertility, menstrual deregulation, and miscarriages [13]. In fact, melatonin sends photoperiodic information that regulates reproductive activity, improving ovarian functions and participating in the follicular development process, including ovulation [14].
Furthermore, being able to rapidly pass through the placenta, melatonin sends photoperiodic information to the fetus, supporting tissue differentiation and hormonal metabolism [15].
From a clinical point of view, it must be borne in mind that endogenous levels of melatonin are reduced with increasing age; this event correlates with cellular senescence, due to the exposition of cells to ROS activity [16]. Aging negatively affects female fertility by lowering oocyte quality, with the increase of aneuploidy [17], and by reducing the follicle and oocyte pool [18]. The main cause of ovarian aging seems to be oxidative stress [19]. So, melatonin exogenous supplementation may also be useful as a treatment for women who are near the end of their reproductive age and want to become pregnant.
In general terms, reproductive disorders triggered through hormonal alterations represent one of the main causes of infertility [20]; this is why many studies have been focused on understanding melatonin functions during pregnancy [21,22].

Molecular Mechanisms of Embryo Implantation
The embryonic implantation is a key and complex process that requires correct communication between blastocyst and uterus. The establishment of uterine receptivity, preimplantation embryo development, and embryo implantation events are mainly regulated by cytokines, chemokines, growth factors, and steroid hormones. Steroid hormones, especially estrogen and progesterone (P4), play important roles in supporting endometrial preparations to establish endometrial receptivity, and allow the maternal clock to synchronize with the embryonic one [23].
In fact, after ovulation, progesterone production in the ovary allows endometrial differentiation, favoring a receptivity state for the implant [24]. About 24 h after fertilization, the embryo begins cell divisions from which blastomeres derive, increasing the total cell number but not the total volume, thanks to a compaction process. During this phase, the embryo is pushed by oviduct cilia towards the uterus, and the internal cells acquire a defined polarity, while the external cells remain apolar and starts to dwindle. From this differentiation process, the blastocyst is derived; it is characterized by an internal cell mass (ICM) and trophectoderm (TE). In the uterus, the blastocyst remains adherent, and for 48 h increases its dimensions. The TE cells, positioned at the external limit of the embryo, are responsible for the endometrial connection. Subsequently, TE cells secrete enzymes to penetrate the endometrium, starting syncytiotrophoblast formation. Thereafter, trophectoderm forms several embryonic structures from which the placenta will derive. With regard to the development of the fetus, ICM cells will be responsible of all embryonic and fetal tissues [25].
In vivo studies in mice [26] have identified a probable embryo receptivity window of about 4 days, which occurs about 7-11 days after the luteinizing hormone (LH) peak. One of the main estrogen mediators responsible for implantation seems to be leukemia inhibitory factor (LIF), an interleukin-6 family member, regulated by p53 [27], that guarantees the uterine receptivity in the implant ( Figure 1). Indeed, scientific studies have reported implant failure in mice when this factor was suppressed.
In molecular terms, endometrial receptivity is an accurate process involving the acquisition of adhesion ligands and receptors. Among the actors involved in this series of events is αVβ3, an integrin present and up-regulated on the apical surface of luminal endometrial cells during implantation, even if its embryonic ligand still hasn't been detected [28]. Another molecule responsible for the regulation of this process is mucin (MUC1), which is present on the endometrial cell surface and is characterized by anti-adhesive properties [29]. Considering the MUC1 activity relative to implantation, it is clear that a local action is needed to remove this barrier, as reported in some studies, where MUC1 down-regulation in endometrial cells was observed in the embryo paracrine action in rabbits [30] (Figure 1).
The existence of conflicting mechanisms allows us to set the timing of the implantation process. Thanks to these molecular events, implantation can be divided into three finely regulated stages: apposition, adhesion, and penetration. These three phases are completed in the "implant window", a period in which the endometrium is receptive to the blastocyst [31].
The pre-implantation stage, known as apposition, was observed in rodent studies [32]. During apposition, TE cells are in close adherence with the endometrial epithelial wall [33]. This close apposition would seem to support the correct embryonic poles orientation, which is critical for placental development [32]. At the molecular level, the pre-implantation stage has been described with the identification of different adhesion molecules, such as αVβ1 integrins and the intracellular adhesion molecule (ICAM-I) [34] (Figure 1).
After this phase, the trophoblast and the luminal epithelium establish a strong bond (adhesion). Embryonic adhesion would seem to be modulated by microenvironmental signals which cause up-regulation in adhesion proteins [35]. Once adhesion has occurred, the blastocyst is inserted into epithelial tissue, and switches into stroma to connect to the maternal vascular system [36]. During this phase, the blastocyst cells begin to activate the matrix metalloproteinase family members, which allow proteolytic digestion of the maternal stromal extracellular matrix, favoring penetration and implantation.
In recent years, the identification of the implantation and development mechanisms has allowed us to understand this process, improving also in vitro fertilization techniques. Despite this, so far only 10-15% of embryos implanted after in vitro fertilization have a positive outcome [37]. To clarify these data, it is important to highlight what factors are involved. Among these, ROS overproduction is, undoubtedly, one of the main factors, being responsible for embryonic damage [38] through the induction of apoptosis [39].

Melatonin Functions on Oocyte Quality and Embryo Implantation
The role of melatonin in human reproduction has been deeply investigated through direct actions on the ovary, supporting the development of good quality oocytes and embryos. In fact, when injected in a systemic way, melatonin was found in higher concentrations in ovary tissue, confirming a targeted action. From the pineal gland, by circadian cyclical release, melatonin mediates the down-regulation of gonadotropin-releasing hormone (GnRH) in a cyclic time [40], assuming a central role in the reproductive system.
Normally, melatonin levels positively correlate with follicular growth, assuming an important role in the ovulatory phase [41]. Melatonin plays an important role as a regulator of several phases of ovulation, as demonstrated by the presence of melatonin binding sites in the membrane fraction of human granulosa cells (GCs), and of melatonin membrane receptors (MT1 and MT2) in granulosa (GC)/luteal cells. In this site, melatonin acts like natural guardian of primordial follicle pool. Its protective action doesn't allow phosphorylation of several important molecular factors. In their work, Jang et al. display how melatonin blocks the activation of extracellular signal-regulated kinase (ERK), protein kinase B (Akt), phosphatidylinositol (3,4,5)-trisphosphate (PIP3), glycogen synthase kinase 3 beta (GSK3β), Forkhead box O3 (FOXO3a), and phosphatase and tensin homolog (PTEN) [42]. The phosphorylation block of PTEN inhibits the PI3K pathway, as well as Akt activation and its down-stream effectors (ERK, GSK3β, FOXO3a) [43].
Another actor in the implantation process is the immune system. During follicle rupture, immune system cells release prostaglandins and cytokines to increase the capillary permeability. This important process leads to an elevated production of free radicals and ROS [44].
High levels of melatonin were found in human pre-ovulatory follicular fluid [45], reducing oxidative stress in the follicles and protecting the oocytes from damage caused by free radicals [46]. Three-fold higher levels of melatonin were found in human preovulatory follicular fluid (FF) than serum levels. Moreover, melatonin concentrations were higher in the fluid of large follicles than in the fluid of small follicles in patients undergoing in vitro fertilization (IVF)-embryo transfer, confirming its fundamental role in this context. In follicular fluid, melatonin does not occur exclusively from its uptake from blood, but could be synthetized by ovarian cells, such as cumulus cells [47]. In this way, melatonin functions can be improved in the ovaries, leading to local specific actions. This melatonin supplement is necessary to reduce oxidative stress in follicles triggered by inflammation-like processes.
Another important data is the mechanism of selection of the ovulatory follicle that seems to be linked with the mRNA expression encoding LH receptors in GCs. In this regard, melatonin treatment increases the mRNA expression of LH receptors in human GCs, highlighting the important role of this molecule in the female fertility. Moreover, melatonin can influence sex steroid production at different phases of ovarian follicular maturation, increasing progesterone production in preantral follicles, as demonstrated in mice by Adriaens et al. [48] Furthermore, an interesting hypothesis views melatonin as an important factor in the seasonal variability of human fertility. Indeed, for all human populations, a seasonality in birth rapports was reported [49]; this variability in fertilization rates, and in the embryo quality, might depend on changes in melatonin secretion, as suggested by some studies [50].
In this regard, the in vitro use of melatonin on embryo culture has been shown to cause a reduction of oxidative stress and apoptosis [51]. Indeed, scientific studies have reported that melatonin is able to increase the number of blastocyst cells, increase GSH levels, and reduce oxidative stress and apoptosis [52], having a protective effect on embryos.
These effects may also derive from actions on the oocyte itself: the early stages of embryogenesis are specifically subject to information contained at the oocyte level. High quality oocytes give rise to well-developed embryos. In particular, during the second meiotic division, the oocyte accumulates oxidative stress, which must be reduced to obtain a good quality embryo [53].
In addition to the direct antioxidant activity, melatonin-induced functions are directly mediated by binding to melatonin membrane receptors MT1 and MT2 [54]. Following stimulation, melatonin receptors activate several signaling pathways involved in embryo implantation: the MT1, MT2, p53, and LIF levels in the uterus were evaluated in order to study the direct influence of melatonin during the implantation period [55]. It has been shown that the differential expression of MT1 and MT2 receptors in pregnant and non-pregnant human uteri is able to influence the cyclic rhythm of myo-endometrial contractility [41].
Studies performed in a mouse culture system displayed that melatonin may be involved in metabolism to promote both the quality and the quantity of embryo development, and that it stimulates the formation of blastocysts during embryogenesis [56].
In vivo studies in mice have reported the ability of melatonin treatment to increase estradiol levels (E2), favoring implantation, but reducing the uterine receptivity period [57]. Similarly, treatment has improved the uterine microenvironment, promoting antioxidant enzymes expression, such as SOD, and catalase (CAT) [58].
From a molecular point of view, melatonin regulates the functionality of p53 by inducing p38-dependent phosphorylation of p53 itself [59]. It was then found that the p53-dependent DNA damage response activation is mediated by MT1 and MT2 in mice [60]. Furthermore, recent research has also shown that MT2 is more up-regulated, highlighting its dominant role in mediating the melatonin action in reproduction. The p21 up-regulation increased p38 activation, favoring p53 phosphorylation and activation, together with its up-regulation [61] (Figure 1). These results indicate that p53 might be a downstream element of MT1-MT2 activation, lastly regulating LIF expression, thereby influencing positively the embryo implantation [62].
Considering that during the pre-implantation period, oxidative stress negatively influences embryo quality and pregnancy success [63], these observations led to the use of melatonin as an adjuvant in in vitro embryo cultures, increasing the embryonic development and blastocyst rate in mice [64].
In this regard, blastocysts treated with melatonin display a significantly higher number of ICM and TE cells, improving intracellular ROS levels [65]. During in vitro culture, embryos are exposed to higher levels of oxidative stress than in vivo [66]. The cause of the increased susceptibility to oxidative stress of mammalian embryos is a high concentration of lipids [67]. Therefore, ROS increase causes loss of membrane integrity and alteration in functional structures [68]. Melatonin improves embryo quality through increasing expression levels of antioxidant genes, reducing mitochondrial damage and apoptosis. Studies on rats confirm melatonin's ability to improve levels of catalase (CAT) and superoxide dismutase (SOD) if exposed to oxidative stress induced by sodium fluoride (NaF), counteracting ROS activity on embryos [69] (Figure 2). In addition, several studies have reported that melatonin is able to regulate apoptotic processes. In particular, according to the concentration of melatonin used, either this molecule can up-regulate the anti-apoptotic gene bcl-2 and down-regulate pro-apoptotic bax and caspase-3 genes [70], reducing apoptosis of embryonic cells and positively impacting mouse embryo cleavage rates (with concentrations of melatonin of 10 and 100 nM), or, in high concentrations (100 µM), can mediate pro-oxidant and pro-apoptotic actions, causing a delay in embryonic development and implantation speed (Figure 2). This last pro-oxidant activity was also reported in the study of Buyukavci et al., where the counteracting melatonin activity on leukemia cell growth is displayed [71]. For this reason, the therapeutic use of melatonin is promising; however, its dosage, especially in pregnancy, requires specific attentions and considerations.
The cytoprotective capacity of melatonin at the ovarian district can delay the aging effects and age-related diseases [72]. This ability of melatonin was confirmed by a recent study that has displayed how the telomere reduction was decreased with melatonin administration thanks to sirtuin 1 (SIRT1) up-regulation [73]. At the molecular level, ROS influences both the telomere length, protective structures located at the 3 end of chromosomes [74], and the sirtuin activity [75]. The sirtuin family plays different roles in cell differentiation, senescence, and apoptosis regulation [76]. The sirtuins expressed in the ovary (SIRT1, SIRT3 and SIRT6) are markers of ovarian aging, as they are positively correlated with the follicular reserve [77] (Figure 2). Indeed, the SIRT1 activity regulates the Eukaryotic Initiation Factor 2 (eIF2) phosphorylation [78], which has been found to be inactive in patients with neurodegenerative diseases, such as Parkinson's and Alzheimer's disease [79].
A further confirmation of the importance of melatonin for the development of the embryo is related to its gene expression activity. In some studies, the increase of the blastocyst quality was reflected in a significant increase in pregnancy rates and observed birth rates [80]. Promoting TE and ICM cells proliferation has yielded a positive effect on cleavage rates. In fact, TE cell reduction causes embryo mortality and implant failure [81], while a low number of ICM cells displays a higher probability of fetal loss or damage in development [82]. The success of embryonic implantation depends on the epidermal receptor expression of ErbB growth factors (ErbB1, ErbB2, ErbB3, ErbB4) in the blastocyst trophectoderm, and on their interaction with the ligands present on the receptive endometrial cells. ErbB1 and erbB4 are the first genes expressed on pre-implant blastocyst [83]. Several studies display an increase in the gene expression of erbB1, pra, p53, and mt2 in mice treated with melatonin [84]. The signaling pathway begins from the heparin binding epidermal growth factor (HB-EGF), an early molecular marker, synthesized in the uterus, and adherent to blastocysts through ErbB1/4. In vitro embryo production alters the expression of these receptors, as they are subjected to environmental stress [85]. Pre-treatment with melatonin in mice subjected to In Vitro Fertilization and Embryo Transfer (IVFET) highlighted the increase of HB-EGF expression in the endometrium [86], increasing the implantation probability [84]. Thus, melatonin administration, reducing oxidative stress, promotes the up-regulation of this factor, favoring the implantation [87].
Clinically, melatonin supplementation was added in IVF protocols, and the effects of this intervention were investigated by clinical trials, some of them randomized and controlled. Considering the proven positive effects of the supplementation of myo-inositol plus folic acid on the follicular fluid composition, and consequently on the oocyte quality, Rizzo et al. examined whether the addition of melatonin supplemented continuously from the day of GnRH administration further improved IVF outcomes. Results showed that the supplementation of melatonin is effective in ameliorating the activity of myo-inositol by improving both oocyte quality and pregnancy outcome in patients with low oocyte quality history [88]. Similarly, Unfer et al. demonstrated that the treatment with myo-inositol and melatonin improves ovarian stimulation protocols and pregnancy outcomes in infertile women with poor oocyte quality [89]. Furthermore, the addition of melatonin to myo-inositol and folic acid significantly improved both oocytes and embryo quality in Poly-cystic Ovary Syndrome (PCOS) women who underwent IVF procedures, thus highlighting a synergistic action between the two substances [90].
Overall, both experimental and clinical studies support the positive effects of melatonin on oocyte quality and embryo implantation, and patients with subfertility or infertility may benefit form melatonin supplementation. Therefore, despite the fact that additional randomized control trials (RCTs) are necessary to recommend the use of melatonin as a first line treatment for patients with poor fertility, clinicians should consider its supplementation as a safe and effective approach to increasing the chance of a successful pregnancy.

Melatonin Functions on Pregnancy Outcomes
The pineal gland, a site of melatonin synthesis and secretion, becomes mature after birth. Therefore, assuming the role of melatonin in pregnancy, it is evident that a strong connection exists between maternal secretion and normal fetal and embryonic development.
Melatonin treatment has been studied in different conditions and during the gestational period. Several studies have shown that melatonin administration from the first IVFET cycles, continued during pregnancy, has been associated with an improvement in pregnancy results [88].
As mentioned previously, melatonin can pass all biological barriers, including the placental one [15]. The passage of maternal melatonin exposes the fetus to a daily rhythm, with low concentrations during the day and high ones during the night, establishing the circadian rhythm [91]. The circadian rhythm is also induced in the fetal organs [92]. This is evidenced through the fetal heart frequency, which is synchronized with maternal activity (i.e., day-night), together with hormonal secretion and physique temperature rhythms [93]. These rhythms are interrupted if the mothers are kept in a continuous luminous environment until the end of the gestation period.
In vivo studies, in which mice were exposed to continuous light during the second half of gestation, contributed different results. First, there was delayed intrauterine growth, and second, the clock genes and steroidogenic genes expression was down-regulated in fetal adrenal, lowering corticosterone production. In this context, the importance of melatonin is evident, considering that with a daily dose administration of this molecule to the mother, a reverse of these alterations is achieved [94].
At the human fetal level, melatonin receptors are expressed in the central nervous system (CNS) and in several areas of the fetal brain [95]. At the molecular level, the binding of melatonin to the MT1 receptor [96] modulates the self-regulating interaction of clock genes, including bmal-1, per1-3, cry1-2, and clock, and their gene products [97]. These participate in circadian rhythm generation and maintenance through transcriptional feedback circuits [98]. The clock gene, constitutively expressed in the suprachiasmatic nucleus (SCN), and bmal-1 gene, expressed cyclically, activates per1-3 and cry1-2 genes transcription. These accumulate in the nucleus, establishing complexes where, by negative feedback, they regulate clock and bmal-1 genes transcription [99].
Melatonin receptors are also present in human peripheral tissues, highlighting how melatonin can also perform other functions [100]. Recent research shows that melatonin stimulates fetal adrenal growth [96], but inhibits cortisol synthesis in adrenal gland [101]. Furthermore, melatonin plays an essential role in influencing fetal gonads [102] and post-natal reproductive development [103]. Moreover, in the trophoblastic cells that constitute the placenta, the "clock genes" are expressed, and control the activated transcriptional feedback. The breaking of this coordinated process can compromise the protective function, with a consequent chain-reaction effect to the immune system [104].
Reporting the melatonin activity on placental tissue can be very useful in understanding the importance of this molecule for the fetus. Placental tissue is characterized by a very strong relationship between its two components, cytotrophoblast and syncytiotrophoblast, and plays a key role during implantation and throughout pregnancy. Recently, several studies reported melatonin activity at local levels and its production by the cytotrophoblast [105]. The role of melatonin at this level was also confirmed by the presence of melatonin membrane receptors both in the cytotrophoblast and in the syncytiotrophoblast. All these findings allowed us to formulate the hypothesis of paracrine and autocrine actions [106]. It is already known that melatonin can induce or block the apoptosis (see above) in cancer cells [107]. A balance between the formation of the syncytiotrophoblast from cytotrophoblasts and its degeneration via apoptosis is necessary to prevent pathologies from developing in the placenta. Melatonin, which, as pointed about above, is produced in cytotrophoblasts [105,106], has a prominent regulatory effect on apoptosis. It has been repeatedly demonstrated that melatonin exhibits anti-apoptotic actions in normal cells, while being pro-apoptotic in cancerous cells [107]. These dual functions are believed to be exploited by the placenta to maintain a balance between the villous cytotrophoblasts and the syncytiotrophoblast [108].
Considering that melatonin receptors are present at the fetal level, there is the evidence that melatonin is involved in neurodevelopment, i.e., developing fetal sleep patterns [109]. This displays how the action of melatonin on the human fetus is not limited to maintaining the circadian rhythm. In fact, during the last trimester of pregnancy, a more rapid development of the fetal brain is closely associated with Rapid Eye Movement (REM) sleep [110]. Melatonin might be one of the factors that regulates both the fetal sleep cycle REM and non-REM sleep [111].
The neuroprotective activity of melatonin, both in the fetal brain and in the adult, is fundamental for optimal development. This effect, and its effectiveness, have been reported in some studies on several animal models, in which the melatonin administration to the mother caused a significative reduction of fetal hypoxia, improving neurodevelopment and decreasing of brain lesions and oxidative stress in the fetus [112]. Tamura et al. proposed that deficient pineal melatonin production may be casually related to the development of spontaneous abortion in cases where chromosomal anomalies and/or uterine abnormalities have been excluded. Recurrent pregnancy loss has been associated with oxidative damage and immunological imbalance; in this context, melatonin acts as a direct powerful free-radical scavenger with immunomodulatory effects. Furthermore, melatonin stimulates the secretion of progesterone, and inhibits the synthesis of prostaglandins which are potent inducers of miscarriage and preterm labor [113].
An emerging concept that links environmental conditions during embryonic development with the risk of developing pathologies after birth is fetal programming. As seen previously, the expression of melatonin varies according to the several moments of fetal development, with fluctuations throughout the pregnancy. Modifications of this program, with altered serum levels, have been observed in complicated pregnancies, supporting the hypothesis of an influence on fetal development [114]. These modifications could be a response to environmental conditions, and increase the risk of the development of pathologies in adulthood [115]. At this regard, altered melatonin serum level seems to be related to anomalous placental development. The reason for this is unbalanced epigenetic mechanisms [116] induced by an increased oxidative stress, which is in turn linked with a higher formation of ROS and free radicals. This leads to incorrec methylation, an epigenetic alteration that could induce modifications in the gene expression patterns and phenotype modifications in adulthood [117].
Melatonin could regulate epigenetic mechanisms by nuclear receptors, leading to DNA bending in the oocyte [118]. This hypothesis suggests that melatonin could activate antioxidant enzyme expression by epigenetic mechanisms. Among these enzymes, Nrf2 seems play a crucial role in epigenetic modifications in fetal programming [119]. For this reason, correct melatonin levels during pregnancy are crucial to prevent fetal reprogramming, protecting the fetus from the risk of developing metabolic disease in adulthood Hobson et al. have evaluated the impact of melatonin on women with severe preeclampsia; the obtained data have reported the ability of melatonin to improve maternal endothelial pro-oxidant injury, showing a very safe profile for mothers and their fetuses. Preeclampsia is a major disorder in human pregnancy, which may include potential fetal and maternal complications such as low birth weight, prematurity, and renal failure, Hemolysis, Elevated Liver enzymes, Low Platelet count (HELLP) syndrome, liver failure, and cerebral edema. The pathogenesis of preeclampsia is complex, and involves high placental oxidative stress [120]. A further confirmation that low night-time melatonin levels, as recorded in pregnant women with preeclampsia, resulted significantly lower than normal pregnancies [121]. So, melatonin supplementation should be considered as a potential disease-preventing agent, with the aim of extending pregnancy duration to improve clinical outcomes [120].
Throughout gestation, serum melatonin concentrations in the mother display fluctuations in both physiological and pathological pregnancies [122]. In particular, a significant increase in serum melatonin occurs after 24 weeks from implantation, increasing again after 32 weeks. Finally, after birth, the values return to physiological levels within two days [123]. This expression variability could be explained by the important role played by melatonin as a pregnancy regulators.
Although the mechanism that determines childbirth is not yet fully understood, a seasonal and daily periodicity of human birth has been identified [124]. The photoperiod would seem to be an important factor in controlling birth time. According to this logic, melatonin is the main mediator of this control, as reported [125]. Several studies have displayed an increase in melatonin levels in amniotic fluid during the pre-natal period. The changes in circulating melatonin levels would appear to be a determining factor in the birth times [126].

Conclusions
A large amount of research has focused on melatonin, shedding light on its mechanisms of action, and its functions. From these studies has emerged the key role of melatonin in processes of embryonic development and human reproduction.
Among the physiological activities of this molecule, one of the most important is the modulator of the circadian reproductive maternal rhythm, favoring uterine receptivity, and establishing the day/night cycle in the fetus.
Another fundamental activity of melatonin is the antioxidant function that allows the direct removal of free radicals from the oocyte and the embryo, supporting implantation and proliferation. More and more data confirm the cytoprotective effect, together with the immunomodulatory one, that would seem essential for the success of pregnancy and correct fetal development. Finally, the latest studies attest to a probable protective effect and in the direct development of the nervous system. These results are validated by the fact that in models with alterations and/or reductions in melatonin secretion levels, efficiently desynchronizing the circadian rhythm, there is a higher probability of significant complications in pregnancy, such as abortion, pre-eclampsia, and neonatal neurological disability [127]. In fact, several data show that pregnant women who are often exposed to light during the night, or who perform night work, have increased incidences of complications during pregnancy. These complications might be related to a deficiency of melatonin secretion in these individuals [113].
Currently, melatonin is present as a food supplement and over-the-counter drug, with high availability, and has no acute or chronic adverse effects.
So far, several clinical studies have been performed on pregnant women: melatonin supplementation has shown no risk, but rather, has displayed positive effects. Therefore, all the obtained data confirm the important role of this molecule in supporting physiological pregnancy. In this regard, further studies would be useful to better define the posology of melatonin in order to achieve the most effective responses, both for the mother and the fetus.


Spinal cord neurenteric cyst: clinical and diagnostic findings and long term follow-up in two dogs

To cite this article: Teresa Gagliardo, Daniele Corlazzoli, Marco Rosati, Swan Specchi, Luciano Pisoni, Sara Del Magno, Simona Pappagalli, Greta Galli & Gualtiero Gandini (2018) Spinal cord neurenteric cyst: clinical and diagnostic findings and long term follow-up in two dogs, Veterinary Quarterly, 38:1, 106-111, DOI: 10.1080/01652176.2018.1542515 To link to this article: https://doi.org/10.1080/01652176.2018.1542515

Case 1
A 4-year-old entire female 25 kg Golden Retriever was referred because of 1-month history of an insidious onset and progressive course of hind limb gait abnormalities. The general physical examination was unremarkable. Neurological examination showed mild proprioceptive ataxia of the hind limbs and marked monoparesis of the right hind limb. Postural reactions were reduced in the left hind limb and absent in the right. Segmental spinal reflexes were normal. The clinical findings were consistent with a thoracolumbar (T3-L3) neuroanatomical localization of the lesion.
Magnetic resonance imaging (MRI) of the thoracolumbar vertebral column and spinal cord was performed using a 0.17 T Magnet (Scan-MR; Esaote, Genova, Italy). Sequences included T1-weighted (T1-W) and T2-weighted (T2-W) images in transverse and sagittal planes. In addition, T1-W images in sagittal, transverse, and dorsal planes were obtained after intravenous administration of paramagnetic contrast media gadoversetamide (Optimark, 0.5 mmol/ml, Mallinckrodt, Dublin, Ireland) at 0.1 mmol/kg bodyweight (BW). MRI showed, at the level of the 13th thoracic vertebra, a lesion lateralized on the right side hyperintense on T2-W, iso-to slightly hypointense on T1-W images, with a mild peripheral contrast enhancement. The abnormal findings were consistent with a roundish, well-demarcated intradural-extramedullary spinal cord space-occupying lesion of 7 mm in diameter. MRI also showed mild dilatation of the central canal cranial to this mass consistent with mild syringohydromyelia, as illustrated in Figure 1.
Based on the MRI findings, the main differential diagnoses included an inflammatory space-occupying lesion, epidermoid cyst, and, less likely, juvenile neoplasia such as nephroblastoma or primary neuroectodermal tumor.
The dog underwent decompressive surgery under general anesthesia. Premedication was performed using acepromazine (0.02 mg/kg BW IM) and methadone (0.3 mg/kg BW IV), followed by induction with propofol (2 mg/kg BW IV). General anesthesia was maintained with isoflurane and oxygen. Intraoperative analgesia was provided by a constant rate infusion of fentanyl (0.005 mg/kg BW/h IV).
A T13-L1 right hemilaminectomy was performed. Bulging of the dura mater was observed immediately following hemilaminectomy. After durotomy, the lesion appeared as a fluid-filled mass ( Figure 2). The mass was not attached to the spinal cord, so it was removed in toto and submitted for histopathological examination.
All excised tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and eosin, alcian blue, and periodic acid-Schiff (PAS).
Histology showed non-encapsulated, partially cystic mass, consisting of moderately cellular epithelial proliferation. The cells were organized in ducts and occasionally branching tubules of cuboidal to columnar, mono-, or bilayered epithelium with intercalating goblet cells and occasional cilia on the luminal surface ( Figure 3). The stroma presented some clefts and empty cavities lined by flattened mesenchymal and/or possibly epithelial cells. There was moderate anisocytosis, the cytoplasm was granulated and eosinophilic. Epithelial cells, characterized by a finely vacuolated cytoplasm, were frequently observed. A moderate anisokaryosis was seen throughout the cells with mainly round nuclei accompanied by few large oval nuclei. Polynucleosis and anisonucleosis were also frequently observed. Special stains revealed multiple PAS positive accumulations within the epithelial cells. Immunohistochemistry was performed with monoclonal mouse anti-pan-cytokeratin antibody (Clone AE1/AE3) (1:50; Dako Agilent Pathology Solution, Glostrup, Denmark) coupled with polyclonal rabbit anti-mouse IgG antibody horseradish peroxidase (1:200; Dako Agilent Pathology Solution, Glostrup, Denmark) and 3,3 0 -diaminobenzidine (DAB) (Vector Laboratories, Burlingame, CA); with polyclonal rabbit anti-glial fibrillary acidic protein (anti-GFAP) antibody (1:500; Dako Agilent Pathology Solution, Glostrup, Denmark) and polyclonal rabbit anti-S100 antibody (1:400; Dako Agilent  Pathology Solution, Glostrup, Denmark) coupled with biotinylated goat anti-rabbit IgG antibody (1:200; Vector Laboratories, Burlingame, CA); with avidin-biotin complex detection kit (Vectastain detection kit, Vector Laboratories, Burlingame, CA) and DAB (Vector Laboratories, Burlingame, CA) as chromogen.
Immunohistochemistry for cytokeratin was positive throughout the epithelial cells, while GFAP and S100 did not produce significant immunopositivity. Giving its similarities with the respiratory epithelium, a spinal neurenteric cyst (NC) was diagnosed.
Postoperative (PO) analgesia consisted of methadone (0.2 mg/kg BW/4 h IV) for the first day and buprenorphine (0.01 mg/kg BW/6 h IV) for the following 2 days. In addition, antibiotic medication (cephalexin 30 mg/kg BW/12 h PO) was administered for 5 days. The dog was discharged 4 days after surgery with the following medication: tramadol (2 mg/kg, BW/8 h PO), omeprazole (0.75 mg/kg BW/24 h PO), and firocoxib (5 mg/kg BW/24 h PO) for 5 days. Postoperatively, the dog was mildly ataxic in the hind limbs and showed a worsening of the monoparesis in the right hind limb, which improved over the following 2 months.
Seven months postoperatively, on neurological examination, the dog showed only a mild right hind limb monoparesis. MRI was performed at that time and revealed right dorsolateral localized meningeal contrast enhancement at the level of T13-L1 and an adjacent focal flattening of the spinal cord. A surgical scar tissue was suspected (Figure 4). The dog slowly continued to improve and, 3 years after surgery, the owner and the referring veterinarian reported that the clinical signs had stabilized and that a slight right hind limb monoparesis remained.

Case 2
A 1-year-old entire male 27.8 kg mix-breed dog was presented with a history of acute onset of ataxia and paresis of the left hind limb, worsening over the following 10 days. At the first neurological examination, the dog was paraplegic, postural reactions were absent in the hind limbs, and segmental spinal reflexes were normal. Nociception was absent in the left hind limb and markedly reduced in the right hind limb. Neuroanatomical localization was T3-L3 myelopathy. The main differential diagnoses included inflammatory/infective (meningomyelitis of unknown origin; bacterial, fungal, viral, and parasitic myelitis), vascular (ischemic and hemorrhagic myelopathy) and, less likely, intervertebral disc extrusion or neoplastic (for example lymphoma and nephroblastoma) diseases.
MRI of the thoracolumbar vertebral column was performed using a 0.3 T Magnet (AIRIS II, Hitachi Medical System, Milan, Italy). Sequences acquired included T1-W and T2-W images in transverse and sagittal planes, T1-W images in transverse planes after intravenous administration of paramagnetic contrast media gadodiamide (Omniscan, 0.5 mmol/ml, GE Healthcare, Oslo, Norway) at 0.1 mmol/kg BW, fluid attenuated inversion recovery (FLAIR) in sagittal planes and short tau inversion recovery (STIR) in dorsal planes.
MRI showed an apparently intradural-intramedullary spinal cord abnormality at the level of the first lumbar vertebra which was hyperintense on T2-W and STIR images, hypointense on T1-W (Figure 1) and FLAIR and appeared, on T1-W post contrast images, to be surrounded by a hyperintense rim. In addition, cranially to the lesion and to the 7th thoracic vertebra, a dilatation of the central canal consistent with mild syringohydromyelia was detected.
Based on MRI findings, the main differential diagnoses included inflammatory disease such as an abscess, congenital anomaly (epidermoid cyst) or juvenile cystic neoplasia. At this time, the owner declined surgical intervention and the dog was treated with prednisolone (1 mg/kg BW/24 h PO). Three days after the first presentation, a subsequent examination showed worsening of the neurological signs with loss of nociception in both hind limbs and a surgical exploration of the lesion was planned. The dog was premedicated with fentanyl (0.002 mg/ kg BW IV) and induced with propofol (5.5 mg/kg BW IV). General anesthesia was maintained with a total intravenous anesthesia by target-controlled infusion of propofol (20 mg/kg BW/h). During the surgery, analgesia was provided by a constant rate infusion of fentanyl (0.01 mg/kg BW/h IV).
A left-sided hemilaminectomy was performed. The exposed dura appeared swollen. A durotomy was performed revealing a reddish, well encapsulated mass attached to the spinal cord.
The mass was adherent to the spinal cord and, after pia mater resection, was isolated with the aid of microbipolar coagulation and dissected with microsurgical instruments and a surgical microscope (Zeiss nc31, Oberkochen, Germany). All the visible mass was removed, fixed in formalin, and submitted for histopathological examination. The specimen was processed and stained as the case 1.
Histology showed an encapsulated, cystic, and multiloculate tissue with moderately cellular epithelial proliferation embedded in moderate amount of stroma and surrounded by adipocytes. Cells were organized in a stratified cuboidal epithelium with cavities filled by mucoid material. In the same areas, there were ducts and tubules characterized by a ciliated respiratory epithelium. Distributed within the stroma were multiple microhemorrhages. Immunohistochemistry was negative for GFAP and positive for cytokeratin and epithelial cells showed PAS positive intracellular material (Figure 3). Postoperatively analgesia consisted of methadone (0.2 mg/kg BW/4 h IV) for the first day, methadone (0.2 mg/kg BW/6 h IV) for the following 2 days. The patient was treated with antibiotics (Pradofloxacin 5 mg/kg BW, once every 24 h PO) until discharge, 7 days later.
The dog regained nociception in the hind limbs within 2 days after the surgery. One week postoperatively, the patient was presented with an increased tone in the left hind limb, was able to ambulate unassisted showing proprioceptive ataxia and paraparesis with knuckling on the dorsum of the paw in the left hind limb. Two months after surgery, the hind limbs ataxia was less severe, and the dog did not show any more knuckling of the paw in the left hind limb.
Follow-up was obtained by telephone calls to the owner, who reported a slowly progressive continuous improvement. Two years after surgery, the owner sent a video, which showed the dog to be well ambulatory with very mild proprioceptive ataxia in the hind limbs.

Discussion
In human medicine, NCs are rare congenital lesions of the central nervous system commonly found in the spinal cord or, more rarely intracranially in the caudal fossa (Gauden et al. 2012;Chakraborty et al. 2016). NCs result from the incomplete separation of the embryonic notochordal plate and endoderm during the third week of human development (Savage et al. 2010). The histopathological features resemble those of the epithelium of the gastrointestinal or respiratory tract and, for this reason, are described as enteric, endodermal, gastrogenic or bronchogenic cysts (Can et al. 2015).
NC in dogs is an extremely rare finding. To date only two intracranial (in the fourth ventricle) and two spinal NCs have been described (Molin et al. 2014;Ferrand et al. 2015;Alder et al. 2017;Kent et al. 2017).
In human medicine, the clinical signs may have a variable onset. An acute onset is thought to be related to a rapid increase in mucin secretion causing sudden spinal cord compression or due to haemorrhage within the cyst (Hicdonmez and Steinbok 2004). This scenario was likely in the second case described which showed an acute onset of clinical signs and microhaemorrhages within the cyst on pathology and to the authors knowledge this has not been previously described in a dog. The late onset and the progressive worsening over time are likely due to the slow growth rate of the ectopic tissue, as described in our first case and in the other published case studies.
In people, NCs can occur at any level of the spinal cord and are most commonly found in the lower cervical and upper thoracic spinal cord segments (Can et al. 2015). In humans, NCs have an intraduralextramedullary location in 95% of cases, often situated close to the ventral spinal cord. In less than 5% of all reported cases, NCs have an intramedullary localization (Jhawar et al. 2012). The two previous reports in veterinary medicine localized the NCs as extradural in one case and intradural-extramedullary in the other (Ferrand et al. 2015;Alder et al. 2017). In our patients, the cyst was intradural-extramedullary in case 1 and intradural-intramedullary in case 2 (Supplementary Video). An intradural-intramedullary location of an NC has not been previously described.
The MRI features of the NCs here reported, were similar to the previous descriptions consisting mainly in iso-hypointensity on T1-W and hyperintensity on T2-W images (Ferrand et al. 2015;Alder et al. 2017). Our cases, as described in human and previous veterinary cases, showed a mild peripheral contrast enhancement. This is hypothesized to be the result of a chronic inflammatory reaction due to leakage of liquid cyst content after spontaneous periodic tearing of the wall membrane (Molin et al. 2014;Yang et al. 2015).
MRI is the modality of choice for evaluating the lesions of the spinal cord (Dennis 2011). In human medicine, MRI has an excellent sensitivity and a moderate specificity for the diagnosis of intradural spinal lesion (Masciarelli et al 2017). In this cases, low field MRI provided adequate information regarding morphology of the lesion and allowed detection of associated cord changes, such as syringomyelia, but did not permit a precise localization or a definitive diagnosis as to the nature of the lesion. To date, the final diagnosis of NC can only be established after histopathological examination.
In human medicine, the epithelium of NC stains positive for epithelial membrane antigen, cytokeratin, and carcinoembryonic antigen, and negative for characteristic neuroectodermal markers such as GFAP (Gauden et al. 2012). These staining properties and the histopathological findings of respiratory/gastrointestinal epithelia strongly suggest that NCs are endodermal in origin (Zarineh et al. 2009). In our cases, the diagnosis of NC was supported by the positivity for PAS and diffuses cytokeratin immunoreactivity. Based on histopathologic features, human NCs are categorized into three types: type A cysts display a layer of cuboidal or columnar epithelium with or without cilia, on a basement membrane composed of type IV collagen; type B cysts show the same characteristics of type A, but have glands and may include mesenchymal tissue such as muscle, fat, lymphoid tissues, cartilage, and bone; type C cysts display the features of type A and include glial elements (Wilkins and Odom 1976). According to this classification, NCs of this report could be classified as type B cysts.
In humans, the treatment of choice for spinal NCs is complete surgical excision (Garg et al. 2008). Since complete resection is not always possible, other modalities of treatment can be performed including simple aspiration, fenestration, and/or marsupialization of the cyst. However, simple aspiration could lead to recurrence, while subtotal resection could induce adhesions with consequently postoperative neurological deterioration (Menezes and Traynelis, 2006).
In people, the recurrence rate of intramedullary NC after surgery was found to be 23%, which is probably an underestimation due to the lack of long-term follow-up in some reports (Kumar and Nayak 2002). The proliferation of non-resected epithelium as well as accumulation of cerebrospinal fluid (CSF) in the residual cavity of the cyst might lead the cyst to recur (Yang et al. 2015). This latter condition is considered the result of the development of iatrogenic pseudomeningocele, a rare complication of spinal surgery in humans (Macki et al. 2014).
The result of surgical treatment was excellent in both the reported cases. The second dog recovered ambulation despite loss of nociception for more than 48 h before surgery.
The use of the surgical microscope and microbipolar coagulation used in the second dog allowed better field visualization and cyst resection. In human spinal cord surgery, the use of surgical microscope, microsurgical techniques, and rigid attention to hemostasis are standard care for the surgical treatment (Gandhi and German 2013).
Spinal NC should be included in the list of differential diagnoses in young adult dogs with spinal cord dysfunction both with insidious and acute onset. Surgical treatment of spinal NC may lead to excellent results when complete resection is possible. No recurrence of clinical signs was seen in these cases in the period of follow up (greater than 2 years). MRI findings cannot distinguish spinal NCs from other pathologies, including neoplasms, which have a poorer prognosis. For this reason, surgical exploration should be considered in spinal patients with evidence of a suspected intradural lesion.

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Funding
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