The limited value of traditional morphometric features in stromatoporoid taxonomy

The morphological variation of stromatoporoids, which are solitary organisms, is partitioned into its presumably genetic and environmental components. Potentially heritable, environmentally mediated and residual components of morphological variability were estimated in a test set containing Devonian stromatoporoids of the genus Gerronostromaria from southern Poland using analysis of variance. The taxonomic importance of traditional morphometric features is limited, because they are dominated by the intra‐skeletal component of variance. Conventional metrics were therefore replaced by stereological and textural quantities. Both stereological and textural features are dominated by the inter‐skeletal and inter‐locality components of variation and thus may be valuable in taxonomic and environmental studies of stromatoporoids. Statistical analyses of these characters (principal component analysis and cluster analysis) were performed. Of 13 characters considered most useful in taxonomic studies, only five have been used previously in conventional species definitions.     

Quantitative studies of rat carotid body type I cell nerve endings

The results of a stereological and morphometric analysis of rat carotid body type I cell nerve endings are described. 66.9% of endings possessed symmetrical junctions. Of the remaining endings, 3.6% were presynaptic and 26% were postsynaptic to type I cells; 3.6% of endings had a reciprocal configuration. Apart from membrane specialisations, no other ultrastructural criteria were found to distinguish the different types of endings. Ventilation with 100% and 10% oxygen showed that the hypoxic mixture reduced synaptic vesicle concentration in the nerve endings; this effect was independent of the innervation to the carotid body.     

Differential role of actin,clathrin, and dynamin in Fc gamma receptor-mediated endocytosis and phagocytosis

Clustering of macrophage Fc gamma receptors by multimeric immunoglobulin complexes leads to their internalization. Formation of small aggregates leads to endocytosis, whereas large particulate complexes induce phagocytosis. In RAW-264.7 macrophages, Fc gamma receptor endocytosis was found to be dependent on clathrin and dynamin and insensitive to cytochalasin. Clathrin also associates with nascent phagosomes, and earlier observations suggested that it plays an essential role in phagosome formation. However, we find that phagocytosis of IgG-coated large (> or =3 microm) particles was unaffected by inhibition of dynamin or by reducing the expression of clathrin using antisense mRNA but was eliminated by cytochalasin, implying a distinct mechanism dependent on actin assembly. The uptake of smaller particles (< or =1 microm) was only partially blocked by cytochalasin. Remarkably, the cytochalasin-resistant component was also insensitive to dominant-negative dynamin I and to clathrin antisense mRNA, implying the existence of a third internalization mechanism, independent of actin, dynamin, and clathrin. The uptake of small particles occurred by a process distinct from fluid phase pinocytosis, because it was not inhibited by dominant-negative Rab5. The insensitivity of phagocytosis to dominant-negative dynamin I enabled us to test the role of dynamin in phagosomal maturation. Although internalization of receptors from the plasma membrane was virtually eliminated by the K44A and S45N mutants of dynamin I, clearance of transferrin receptors and of CD18 from maturing phagosomes was unaffected by these mutants. This implies that removal of receptors from the phagosomal membrane occurs by a mechanism that is different from the one mediating internalization of the same receptors at the plasma membrane. These results imply that, contrary to prevailing notions, normal dynamin and clathrin function is not required for phagocytosis and reveal the existence of a component of phagocytosis that is independent of actin and Rab5.     

Geometrical characteristics of mitochondria and active presynaptic zones in the dorsal horn of the cat spinal cord

A morphometric electron microscope study was carried out on the ultrastructure of 140 presynaptic terminals (PT) in the dorsal horn of the cat spinal cord. Some spatial characteristics of mitochondria and active zones (AZ) for these PT were examined using statistical stereological analysis techniques. The distribution of 3-dimensional mitochondrial radii was determined, together with average mitochondrial volume, mean area of the external membrane, and average numbers of the test population falling within the PT. Distribution of diameters and estimates of mean area of AZ were obtained, as well as of mean value of synaptic clefts. The relationship between findings from morphometric research and parameters of processes underlying ionic transmembrane diffusion and accumulation is discussed.Dnepopetrovsk State University. A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 741–747, November–December, 1989.     

Effect of phagocytosis on receptor distribution and endocytic activity in macrophages

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or IgG-coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, alpha 2-macroglobulin X protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage-like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor-mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated ligand uptake.     

Genetic similarity between the South Atlantic and the western North Pacific Maurolicus (Stomiiformes: Actinopterygii) taxa,M. walvisensis Parin & Kobyliansky and M. japonicus Ishikawa: evidence for synonymy?

An analysis of the 524 nucleotide long segment in the 16SrDNA mitochondrial gene of Maurolicus japonicus revealed that there are two major haplotypes (E and H) and two minor haplotypes (I and J), which comprise 63·6, 27·2, 4·6 and 4·6% of the population, respectively. The nucleotide sequence of the major haplotype E is identical to that of the most common haplotype in Maurolicus walvisensis (63·6% of the population). The other haplotypes of M. japonicus are almost identical to that of the haplotype E with only a single (haplotypes I and J) or three nucleotide differences (haplotype H). Phylogenetic trees of all the 16SrDNA haplotypes found thus far in the Maurolicus taxa show that the relationships among the haplotypes of M. japonicus and M. walvisensis are indistinguishable but that they are clearly distinctive from those of Maurolicus muelleri . Examination of the morphometric characteristics of specimens reveals similarities among the individuals of different haplotypes of M. japonicus and also between M. japonicus and M. walvisensis in almost all characteristics. The results suggest that despite the current ocean-wide allopatric distribution between M. japonicus and M. walvisensis , the two taxa are conspecific as M. japonicus .     

Polymorphonuclear cell derangements in type I diabetes

Polymorphonuclear cell function had been studied in 58 Type I diabetic subjects. Chemotaxis, phagocytosis, adherence, bactericidal activity, NBT reduction capacity were evaluated. We enumerated gamma Fc receptor bearing polymorphonuclear cells and the percentage of immune complexes containing polymorphonuclear cells. These data were studied in accordance with glycemic levels and the presence of infections. All polymorphonuclear functions were decreased compared to non-diabetic subjects with the exception of phagocytosis. The efficiency of the diabetic sera on normal subjects polymorphonuclear cells was decreased (bactericidal activity, chemotactic index and phagocytosis). These abnormalities were independent of the presence of infection. No correlation was found with glycemic level. The percentage of cells bearing an Fc gamma receptor was less in diabetics than in normal (70.1 +/- 17.4 vs 80.2 +/- 7.8%). The percentage of immune complexes containing polymorphonuclear cells was increased (n = 16, 9.06 +/- 4.7 vs 4.75 +/- 2.1% in normals). There again, these data are without correlation with the presence of infections or glycemic level.     

A morphometric analysis of cytological features of tall cell variant and classical papillary carcinoma of the thyroid

In order to assess whether morphometric parameters could be of value in distinguishing between tall cell variant and classical pattern of thyroid papillary carcinoma, the fine needle aspiration cytology (FNAC) samples of 14 cases were analysed using Arcimage 5 software on an Acorn computer. Histological examination of the specimens allowe classification of nine of them as classical pattern and the remaining five as tall cell variants. The nuclear diameter (NDD) and standard deviation distribution (NDSDD), th nuclear area (NAD) and standard deviation distribution (NASDD), and the nuclear/cytoplasmic ratio (NCR) were assessed on May-Grunwald-Giemsa stained smears. Statistical analysis was performed by use of one-way analysis of variance (ANOVA) of the two groups as identified by histology. Whilst NDD (P = 0.007), NAD (P = 0.015) and NADSD (P = 0.026) all appeared statistically significant, NDSD (P = 0.06) and NCR (P = 0.71) were not. The cytological diagnosis of papillary carcinoma is established and reproducible, but morphometric data on the thyroid have so far focused on the differential diagnosis between benign and malignant nodules. The choice of simple morphometric parameters appears to be helpful in the preoperative distinction between the classical pattern and tall cell variant of papillary carcinoma.     

Serotonin-containing epithelial cells in rat duodenum. I. Quantitative morphometric study of the distribution density

Serotonin (5HT)-containing epithelial cells in rat duodenum were studied quantitatively by three-dimensional morphometric analysis. Longitudinal sections covering the whole length of rat duodenum were stained by either 5HT immunohistochemistry or by glyoxylic acid fluorescent histochemistry. Three-dimensional values for positive cell density, namely the number of 5HT cells per unit volume of the epithelium, were obtained by stereological morphometry with the aid of a computer-assisted image analyzer. This analytical method provides an absolute value for the distribution density of 5HT-containing cells regardless of thickness of sections, or which of the two histochemical procedures is used. The mean number of such cells per unit volume was higher in the crypts than in the villi but varied little along the duodenum. The density of 5HT cells in a given duodenal region, however, varied greatly among individual animals. The villi of the 10 to 16-mm segment from the pylorus were identified as having the smallest individual variation and therefore as being the most suitable for statistical evaluation in future pharmacohistochemical investigations.     

Serotonin-containing epithelial cells in rat duodenum. I. Quantitative morphometric study of the distribution density

Summary Serotonin (5HT)-containing epithelial cells in rat duodenum were studied quantitatively by three-dimensional morphometric analysis. Longitudinal sections covering the whole length of rat duodenum were stained by either 5HT immunohistochemistry or by glyoxylic acid fluorescent histochemistry. Three-dimensional values for positive cell density, namely the number of 5HT cells per unit volume of the epithelium, were obtained by stereological morphometry with the aid of a computer-assisted image analyzer. This analytical method provides an absolute value for the distribution density of 5HT-containing cells regardless of thickness of sections, or which of the two histochemical procedures is used. The mean number of such cells per unit volume was higher in the crypts than in the villi but varied little along the duodenum. The density of 5HT cells in a given duodenal region, however, varied greatly among individual animals. The villi of the 10 to 16-mm segment from the pylorus were identified as having the smallest individual variation and therefore as being the most suitable for statistical evaluation in future pharmacohistochemical investigations.     

Intraspecific taxonomic differentiation in ticks (Acari: Ixodidae) in the view of the morphological conception of species

The criterions for two most used intraspecific taxonomic ranks of ticks--subspecies and morphotype, have been formulated on the basis of the study of morphological variation in the distribution range of all active ontogenetic stages of 11 polymorphic species. All these species are vectors of transmissible diseases. They have vast distribution ranges and different types of host-parasite relationships. Subspecies have the complexes of visual morphological differences expressed in one or both sexes of mature ticks more limited, than those of related species. At immature stages differences of subspecies consist more often in morphometric characters and can be established by the methods of mathematical statistics only. Morphotypes, as a rule, differ at each corresponding stage by "their own" complexes of morphometric characters. All differential parameters of studied morphotypes are overlapped, but have statistically significant differences (by the Student's test). The concrete variations of differentiation by subspecies and morphotypes have been considered. The historical factors of intraspecific differentiation have been reconstructed for each species.     

Phylogenetic lineage and pilus protein Spb1/SAN1518 affect opsonin-independent phagocytosis and intracellular survival of Group B Streptococcus

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.     

Green plant photosystem I binds light-harvesting complex I on one side of the complex

We report a structural characterization by electron microscopy of green plant photosystem I solubilized by the mild detergent n-dodecyl-alpha-D-maltoside. It is shown by immunoblotting that the isolated complexes contain all photosystem I core proteins and all peripheral light-harvesting proteins. The electron microscopic analysis is based on a large data set of 14 000 negatively stained single-particle projections and reveals that most of the complexes are oval-shaped monomers. The monomers have a tendency to associate into artificial dimers, trimers, and tetramers in which the monomers are oppositely oriented. Classification of the dimeric complexes suggests that some of the monomers lack a part of the peripheral antenna. On the basis of a comparison with projections from trimeric photosystem I complexes from cyanobacteria, we conclude that light-harvesting complex I only binds to the core complex at the side of the photosystem I F/J subunits and does not cause structural hindrances for the type of trimerization observed in cyanobacterial photosystem I.     

Molecular architecture of the inner membrane of mitochondria from rat liver: a combined biochemical and stereological study

The molecular structure of mitochondria and their inner membrane has been studied using a combined approach of stereology and biochemistry. The amount of mitochondrial structures (volume, number, surface area of inner membrane) in a purified preparation of mitochondria from rat liver was estimated by stereological procedures. In the same preparation, the oxidative activity of the respiratory chain with different substrates and the concentration of the redox complexes were measured by biochemical means. By relating the stereological and biochemical data, it was estimated that the individual mitochondrion isolated from rat liver has a volume of 0.27 micron 3, an inner membrane area of 6.5 micron 2, and contains between 2,600 (complex I) and 15,600 (aa3) redox complexes which produce an electron flow of over 100,000 electrons per second with pyruvate as substrate. The individual redox complexes and the H+-ATPase together occur at a density of approximately 7,500/micron 2 and occupy approximately 40% of the inner membrane area. From the respective densities it was concluded that the mean nearest distance between reaction partners is small enough (70-200 A) to cause the formation of micro-aggregates. The meaning of these results for the mechanism of mitochondrial energy transduction is discussed.     

Structural and functional study of acidification in the process of phagocytosis

Morphometry and cytofluorometry of the kinetics of phagocytosis were applied to study the quantitative mechanisms of acidification in the area of contact between the plasma membrane of macrophages and Candida albicans yeast cells conjugated with fluorescein isothiocyanate. It was found by stereological transformation of the morphometry data that the main part of macrophages phagocytose the limiting amount of particles by minute 5-10. A good agreement was established between the cytofluorometric histograms and the stereological data. This made it possible to evaluate, using the calibration curve, the pH on the surface of the phagocytosed material based on the fluorescence quenching. As an advantage of the suggested comprehensive approach to the study of acidification, the authors stress the possibility of making structural-functional analysis of the rearrangements occurring in the intact phagocytosing cell.     

Class III polyhydroxybutyrate synthase: involvement in chain termination and reinitiation

Polyhydroxybutyrate (PHB) synthase catalyzes the polymerization of (R)-3-hydroxybutyryl-CoA (CoA = coenzyme A) into high molecular weight PHB. Recombinant wild-type (wt) class III synthase from Allochromatium vinosum (PhaCPhaE(Av)), antibodies to this synthase and to PHB, and [(14)C]hydroxybutyryl-CoA (HB-CoA) have been used to detect oligomeric hydroxybutyrate (HB) units covalently bound to the synthase using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Although a distribution of products is typically observed, short (HB)(n)-bound synthases (designated species I) are most prevalent at low substrate to enzyme (S/E) ratios. Species I is similar to (HB)(n)-PhaC(Av) (n = 3-10 at minimum) recently identified using D302A-PhaCPhaE(Av) (Tian, J., Sinskey, A. J., and Stubbe, J. (2005) Biochemistry 44, 1495-1503). Species I is shown to be an intermediate in the elongation process of PHB synthesis in vitro. The reaction catalyzed by the wt synthase in vitro was further studied under two sets of conditions: at high (70000) and low (<200) S/E ratios. At high S/E ratios, kinetic analysis of the reaction of HB-CoA with the wt synthase monitored using antibodies to PhaCPhaE(Av) and Western blotting revealed the disappearance of PhaC(Av) at early time points and its reappearance as the molecular weight of the PHB approached 1.8 MDa. At low S/E ratios, species I was observed to increase with time after complete consumption of all of the HB-CoA. The results from studies under both sets of conditions suggest that an inherent property of the synthase is chain termination and reinitiation.     

The coleoptile chloroplast: Distinct distribution of xanthophyll cycle pigments,and enrichment in Photosystem II

Recent studies have shown that coleoptile chloroplasts operate the xanthophyll cycle, and that their zeaxanthin concentration co-varies with their sensitivity to blue light. The present study characterized the distribution of photosynthetic pigments in thylakoid pigment–protein complexes from dark-adapted and light-treated coleoptile and mesophyll chloroplasts, the low temperature fluorescence emission spectra, and the rates of PS I and PS II electron transport in both types of chloroplasts from 5-day-old corn seedlings. Pigments were extracted from isolated PS I holocomplex, LHC IIb trimeric and LHC II monomeric complexes and analyzed by HPLC. Chlorophyll distribution in coleoptile thylakoids showed 31% of the total collected Chl in PS I and 65% in the light harvesting complexes of PS II. In mesophyll thylakoids, the values were 44% and 54%, respectively. Mesophyll and coleoptile PS I holocomplexes differed in their Chl t a/Chl t b ratios (8.1 and 6.1, respectively) and -carotene content. In contrast, mesophyll and coleoptile LHC IIb trimers and LHC II monomers had similar Chl t a/Chl t b ratios and -carotene content. The three analyzed pigment–protein complexes from dark-adapted coleoptile chloroplasts contained zeaxanthin, whereas there was no detectable zeaxanthin in the complexes from dark-adapted mesophyll chloroplasts. In both chloroplast types, zeaxanthin and antheraxanthin increased markedly in the three pigment–protein complexes upon illumination, while violaxanthin decreased. In mesophyll thylakoids, zeaxanthin distribution as a percentage of the xanthophyll cycle pool was: LHC II monomers > LHC IIb trimers > PS I holocomplex, and in coleoptile thylakoids, it was: LHC IIb trimers > LHC II monomers = PS I holocomplex. Low temperature (77 K) fluorescence emission spectra showed that the 686 nm emission of coleoptile chloroplasts was approximately 50% larger than that of mesophyll chloroplasts when normalized at 734 nm. The pigment and fluorescence analysis data suggest that there is relatively more PS II per PS I and more LHC I per CC I in coleoptile chloroplasts than in mesophyll chloroplasts. Measurements of t in vitro uncoupled photosynthetic electron transport showed approximately 60% higher rates of electron flow through PS II in coleoptile chloroplasts than in mesophyll chloroplasts. Electron transport rates through PS I were similar in both chloroplast types. Thus, when compared to mesophyll chloroplasts, coleoptile chloroplasts have a distinct PS I pigment composition, a distinct chlorophyll distribution between PS I and PS II, a distinct zeaxanthin percentage distribution among thylakoid pigment–protein complexes, a higher PS II-related fluorescence emission, and higher PS II electron transport capacity. These characteristics may be associated with a sensory transducing role of coleoptile chloroplasts.     

Sec22b Is a Negative Regulator of Phagocytosis in Macrophages

The endoplasmic reticulum (ER) is proposed to be a membrane donor for phagosome formation. In support of this, we have previously shown that the expression level of syntaxin 18, an ER-localized SNARE protein, correlates with phagocytosis activity. To obtain further insights into the involvement of the ER in phagocytosis we focused on Sec22b, another ER-localized SNARE protein that is also found on phagosomal membranes. In marked contrast to the effects of syntaxin 18, we report here that phagocytosis was nearly abolished in J774 macrophages stably expressing mVenus-tagged Sec22b, without affecting the cell surface expression of the Fc receptor or other membrane proteins related to phagocytosis. Conversely, the capacity of the parental J774 cells for phagocytosis was increased when endogenous Sec22b expression was suppressed. Domain analyses of Sec22b revealed that the R-SNARE motif, a selective domain for forming a SNARE complex with syntaxin18 and/or D12, was responsible for the inhibition of phagocytosis. These results strongly support the ER-mediated phagocytosis model and indicate that Sec22b is a negative regulator of phagocytosis in macrophages, most likely by regulating the level of free syntaxin 18 and/or D12 at the site of phagocytosis.     

Morphological,cytochemical, functional,and proliferative characteristics of four murine macrophage-like cell lines

The aim of the present study was to obtain objective data on the morphology and quantitative information about other characteristics of murine macrophage-like cell lines J774.1, PU5-1.8, WEHI-3, and P388-D1, and to compare the findings with those in resident and exudate macrophages collected directly from mice. Fetal fibroblasts were included to serve as controls.Evaluation of the morphological data showed that the cell lines J774.1 and WEHI-3 are almost identical in most respects, that the cells of P388-D1 differ widely from both of the former lines, and that the morphometric parameters of cell line PU5-1.8 occupy an intermediate position. The cells of the P388-D1 line show the most similarity to resident and exudate macrophages, and cell lines J774.1 and WEHI-3 the least. Fetal fibroblasts had divergent values for all morphometric parameters. Good correspondence was found when the quantitative data obtained by morphometric analysis of the cells in question were compared with the morphological pictures.No gross differences as to cytochemical characteristics were found between the cells of the four cell lines, except for 5′-nucleotidase activity. The occurrence of IgG receptors and the ingestion of EIgG were also similar, but the percentage of cells with C3b receptors was much lower in two of the cell lines (WEHI-3 and P388-D1) and the level of EIgMC ingestion was very much higher in one (J774.1) compared with both the other cell lines and the resident and exudate macrophages. The ingestion of opsonized bacteria and latex varied widely within and between the cell lines. Quantitative data on the binding of monoclonal antibodies by the cells of the macrophage cell lines and the resident and exudate macrophages showed a wide variation. The doubling time of the cell lines is on average 1 day; distinct differences were found between these lines with respect to the lag-time of proliferation after replating.Cluster analysis and statistical analysis of morphological and other characteristics gave insight into the degree of resemblance between the cells of the four cell lines on the one hand and the resident and exudate macrophages on the other.     

Sperm head morphometry in ejaculates of adult marmosets (Callithrix jacchus): A model for studying sperm subpopulations and among-donor variations

In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P < 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies.