Showing papers in "Molecular Human Reproduction in 1995"
205 citations
TL;DR: The rates of non-disjunction and unbalanced pre-division after > or = 24 h in culture were similar to the rates in fresh oocytes, and an increase of aneuploidy with maternal age was detected, which although slight, was significant.
Abstract: A large proportion of patients undergoing in-vitro fertilization (IVF) are aged > or = 35 years. It has been estimated that in this age group, 50% of embryos are chromosomally abnormal, with aneuploidy being the major contributing factor. Since the origin of most aneuploidies is maternal meiosis I non-disjunction, unfertilized oocytes could be safely screened for aneuploidy by analysing their first polar bodies. To determine the feasibility of first polar body aneuploidy analysis, polar bodies were analyzed by fluorescence in-situ hybridization (FISH) using probes simultaneously for chromosomes X, Y, 18, 13/21 or X, Y, 18 and 16. Within 6 h of retrieval, 88% showed a normal segregation involving a single chromosome of each kind, with double-dotted hybridization signals, corresponding to dyads (chromosomes in metaphase I composed of two chromatids). The rest showed non-disjunction of full dyads (6%), or an unbalanced pre-division of dyads (6%), which gives a segregation of one chromatid or one dyad and a chromatid with the first polar body. But only 34% of polar bodies analysed 24 h after retrieval or later showed a normal segregation, with most of the other polar bodies showing balanced pre-division, with two separated hybridization signals for all the chromosomes analysed. The rates of non-disjunction and unbalanced pre-division after > or = 24 h in culture were similar to the rates in fresh oocytes. When both types of aneuploidy were considered together, an increase of aneuploidy with maternal age was detected, which although slight, was significant (P = 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)
151 citations
TL;DR: Analysis of 211 oocytes from failed in-vitro fertilizations from 58 patient couples resulted in the determination of several previously undetectable phases at which fertilization arrests, which have implications for the diagnosis and treatment of female, as well as male, infertility.
Abstract: The goal of fertilization is the union of one, and only one, sperm nucleus with the female pronucleus within the activated oocyte. For this to occur successfully, several events must transpire, including the incorporation of the entire spermatozoon into the oocyte, the completion of meiotic maturation with the extrusion of the second polar body, the metabolic activation of the previously quiescent oocyte, the decondensation of the sperm nucleus and the maternal chromosomes into the male and female pronuclei respectively, and the cytoplasmic migrations of the pronuclei, which bring them into apposition. Defects in any of these events are lethal to the zygote and might prove to be causes of infertility. In this study, the microtubules and DNA were imaged in inseminated human oocytes that had been discarded as unfertilized. The presence and number of incorporated sperm tails were also documented using a monoclonal antibody specific for the posttranslationally modified acetylated-α-tubulin found in the tail, but not the oocyte, microtubules. An analysis of 211 oocytes from failed in-vitro fertilizations from 58 patient couples resulted in the determination of several previously undetectable phases at which fertilization arrests : (i) metaphase II arrest ; (ii) arrest after the successful incorporation of the spermatozoon ; (iii) arrest after the formation of the sperm aster ; (iv) arrest during mitotic cell cycle progression ; and (v) arrest during meiotic cell cycle progression. Data on polyspermy and arrested embryonic development are also presented. These results have implications for the diagnosis and treatment of female, as well as male, infertility. They also provide a rationale for the reasonable use of intracytoplasmic sperm injection (ICSI) therapy, although they suggest that cases intractable to this approach will be found. Concerns are raised about the use of seemingly 'unfertilized' but inseminated oocytes for subsequent re-inseminations, ICSI or even research, since the fertilization process might have arrested after sperm penetration. These results demonstrate that the proper union of the parental genomes requires a series of cytoskeletal-mediated events on the oocyte surface and within the oocyte proper, and that failure at any phase results in the arrest of human fertilization.
118 citations
TL;DR: The menstrual cycle-dependent expression of IL-6 suggests that this cytokine may play a role in changes in endometrium that prepare this tissue for implantation and menstrual shedding.
Abstract: In order to be prepared for implantation, human endometrium undergoes a predictable series of proliferative and secretory changes. Cytokines play an important role in regulation of these changes. Therefore, in this study, we immunolocalized the cytokine, interleukin-6 (IL-6), its receptor and the signal transducer gp130 in human endometrium throughout the menstrual cycle. During the entire menstrual cycle, the IL-6 receptor and gp130 were found primarily in the endometrial glands and to a lesser extent in the stroma. The immunoreactivity of these proteins did not change in endometrial cells during the entire menstrual cycle with an exception of reduced immunoreactivity of gp130 in endometrial glands during menstrual phase. Immunostaining showed that immunoreactive IL-6 was weakly expressed in human endometrium during the proliferative phase. Strong immunoreactivity for IL-6 appeared in endometrium during the putative 'implantation window'. Expression was by far most pronounced both in the glandular and surface epithelial cells. The amount of immunoreactive IL-6 in the epithelium progressively increased during the secretory/menstrual phases. During the late secretory phase, only stromal cells in the upper functionalis exhibited immunoreactivity for IL-6. Western blot analysis corroborated the immunohistochemical data. Human endometrial IL-6 consisted of a protein with an apparent mobility of 26 kDa. The immunoreactive band of IL-6 was weak in the proliferative phase. The expression of this protein increased progressively during the secretory/menstrual phases. The findings show a cell-specific pattern of distribution for immunoreactive IL-6 in human endometrium. The menstrual cycle-dependent expression of IL-6 suggests that this cytokine may play a role in changes in endometrium that prepare this tissue for implantation and menstrual shedding.
105 citations
TL;DR: The mechanism(s) by which culture medium composition might affect multinucleation of human blastomeres is discussed, as is the significance of these data for reliable preimplantation diagnosis of genetic status.
Abstract: Human embryos were disaggregated into component blastomeres 42-72 h after insemination. The blastomeres were scored for the number of nuclei present and blastomeres of known nuclear morphology were returned to individual culture drops for 16-20 h, after which they were scored for cleavage and nuclear morphology. In all, 48% of mononucleated blastomeres cleaved during this period, but only 76% of these produced two mononucleated daughter blastomeres; in the remainder, one or more of the blastomeres was abnormally nucleated. During overnight culture, 30% of multinucleated blastomeres and 30% of anucleate blastomeres cleaved, the majority producing abnormally nucleated daughter blastomeres. The majority of blastomeres which showed no sign of cleavage after overnight culture retained the same nuclear morphology as when originally disaggregated. However, a small number of mononucleated blastomeres contained two nuclei after culture, indicating that karyokinesis may have taken place in the absence of cytokinesis. Overall, approximately 30% of blastomeres with more than one nucleus seemed to arise by this mechanism, the remainder probably arising by errors of chromosome segregation and/or packaging at mitosis. In addition, 25/111 mononucleated daughter cells arose either after abnormal division of mononucleated parent cells or after division of multinucleated cells, suggesting that approximately 23% of newly formed mononucleated cells might be chromosomally abnormal. The results of DNA quantitation indicated that very few (12/131, 9.2%) blastomeres (whether uni- or multinucleated) had a DNA content outside the 2-4C range. The embryos used for these studies had been cultured in one of three commonly used in-vitro fertilization (IVF) media: modified T6, Earle's balanced salts or Universal IVF medium (a commercial medium from Medi-Cult). A retrospective analysis was carried out of the number of embryos containing multinucleated blastomeres at disaggregation and of the total proportion of isolated blastomeres which were multinucleated in three groups of embryos, each of which had been cultured in one of the IVF media. Both these parameters were found to vary between cohorts of embryos cultured in the different media. The mechanism(s) by which culture medium composition might affect multinucleation of human blastomeres is discussed, as is the significance of these data for reliable preimplantation diagnosis of genetic status.
91 citations
TL;DR: The results suggest an abnormal regulation of HILDA/LIF secretion in such circumstances, and the clinical implication of those data is discussed.
Abstract: Human interleukin for DA cells/leukaemia inhibitory factor (HILDA/LIF) is a cytokine with pleiotropic effects involved in successful murine implantation. We evaluated human uterine HILDA/LIF production by monitoring its in-vitro secretion by endometrial explant cultures obtained from individuals in either normal or pathological conditions. The cytokine secretion was standardized using the day 5:day 1 ratio of HILDA/LIF concentration in supernatants of such cultures, hereby termed HILDA/LIF production index (HLPI). Our results confirmed that HILDA/LIF is secreted by the human endometrium as assessed by secretion at every phase of the cycle in either normal fertile women, or women bearing intrauterine devices. This was also the case for samples obtained from infertile women presenting repeated failures of embryonic implantation or unexplained primary sterility. However, the HLPI were significantly lower in those latter two groups when compared to fertile women. These results suggest an abnormal regulation of HILDA/LIF secretion in such circumstances, and the clinical implication of those data is discussed.
88 citations
TL;DR: The method may appear useful for the detection of oocytes with common chromosomal aneuploidies in IVF patients of advanced maternal age by polar body sampling.
Abstract: Chromosomal aneuploidies contribute considerably to the low pregnancy rate in in-vitro fertilization (IVF). The objective of this experimental work was to explore the possibility of detecting common aneuploidies in oocytes by polar body sampling. The study included 45 infertile patients of advanced maternal age participating in an IVF programme. The first polar body was removed prior to fertilization or both the first and second polar bodies were removed after fertilization and studied by fluorescent in-situ hybridization (FISH) using chromosome-specific probes for chromosomes X, 18 and/or 13/21. Of 155 oocytes with FISH results, 36 demonstrated chromosomal abnormalities. Of 119 oocytes predicted to be free from aneuploidy of chromosomes X, 18 and/or 13/21, 72 were normally fertilized, cleaved and transferred in 23 treatment cycles, which resulted in two healthy deliveries and three ongoing pregnancies confirmed to be unaffected by chorionic villous sampling. The method may appear useful for the detection of oocytes with common chromosomal aneuploidies in IVF patients of advanced maternal age.
84 citations
TL;DR: It appears that LIF may act directly or indirectly, by inducing the expression of other cytokines, to regulate the temporal and spatial production and activity of proteases and protease inhibitors to create a favourable environment for implantation.
Abstract: Several growth factor ligand and receptor gene products have been shown to play roles during preimplantation mammalian development. Genes for insulin-like growth factors (IGFs), transforming growth factors (TGFs), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and receptors for insulin, IGF, PDGF, TGF alpha and epidermal growth factor (EGF) are expressed by early embryos of several species including mouse, rat, cow and sheep. Roles of growth factors during early development have been demonstrated by addition of purified growth factors to culture medium or by molecular genetic techniques that interfere with gene expression. In this way, it has been shown that successful development of the blastocyst is dependent on the action of epidermal growth factor (EGF) and leukaemia inhibitory factor (LIF). Recent experiments show that both LIF and EGF stimulate secretion of urokinase-type plasminogen activator (uPA) and gelatinase B/matrix metalloproteinase-9 (MMP-9) in day 7 mouse blastocyst outgrowths. At the same time, tissue inhibitors of MMPs (TIMPs) are also expressed by embryonic, decidual and uterine tissues during the implantation process. It appears that LIF may act directly or indirectly, by inducing the expression of other cytokines, to regulate the temporal and spatial production and activity of proteases and protease inhibitors to create a favourable environment for implantation.
81 citations
TL;DR: The world's first clinical pregnancy resulting from DNA-based enrichment for X-bearing human spermatozoa, for prevention of X-linked hydrocephalus is reported, and this technique should be widely applicable to gender selection for the prevention of genetic disorders.
Abstract: We report the world's first clinical pregnancy resulting from DNA-based enrichment for X-bearing human spermatozoa, for prevention of X-linked hydrocephalus. Sperm separation was followed by embryo biopsy and nested multiplex polymerase chain reaction (PCR) for gender determination. Enriched populations of X- bearing spermatozoa ranging from 80 to 89% pure as determined by fluorescence in-situ hybridization (FISH) resulted in in-vitro fertilization (IVF) rates indistinguishable from normal IVF procedures (65%). In two separate biopsy procedures, 7/9 and 15/16 of the resulting embryos were determined to be female by multiplex PCR. Embryo transfer resulted in a karyotypically normal female fetus. This technique should be widely applicable to gender selection for the prevention of genetic disorders
70 citations
TL;DR: It is suggested that low concentrations of MBP within the feto-placental unit increase susceptibility to fetal loss, possibly via an infection-induced placental cytokine imbalance.
Abstract: The distribution of mannan binding protein (MBP) in blood donor sera was determined by enzyme-linked immunosorbent assay to establish normal concentrations. Abnormally low MBP concentrations were found in 16% (21 out of 135) of female partners and 14% (15 out of 108) of male partners of couples experiencing recurrent miscarriage, compared with < 5% of obstetrically normal controls (P < 0.005). This relationship was even stronger (9.5 versus 1.0%) and more significant (P < 0.002) when only subjects presumed to be homozygous for the mutant allele responsible for MBP deficiency were considered. By immunohistochemistry, MBP could be demonstrated in first trimester placenta. We suggest that low concentrations of MBP within the feto-placental unit increase susceptibility to fetal loss, possibly via an infection-induced placental cytokine imbalance.
68 citations
TL;DR: A possible autocrine-paracrine role for the IL-1 system in the human endometrium and embryonic implantation is supported, and the regulated presence of the icIL-1ra in the humans' endometrial cells is demonstrated.
Abstract: There is evidence suggesting the importance of the interleukin-1 receptor type I (IL-1Rtl) and interleukin-1 beta (IL-1 beta) as mediator in local intercellular interactions in endometrial tissue and embryonic implantation. To complete our understanding of the entire endometrial IL-1 system in humans, we have investigated the immunohistochemical distribution of IL-1 receptor antagonist (IL-1ra) in the human endometrium throughout the menstrual cycle. We have also identified the forms of IL-1ra present in human endometrial cells. Immunoreactive IL-1ra was found in both cryostat and paraffin-embedded sections of human endometrium using the alkaline phosphatase-peroxidase (A-P) method with two different IL-1ra antibodies. IL-1ra was present throughout the entire menstrual cycle, located primarily in the endometrial epithelium. However, IL-1ra staining was significantly higher during follicular phase in comparison with early and mid-late luteal phases. Reverse transcriptase polymerase chain reaction of cultured stromal and glandular cells showed that these cells express the intracellular form of IL-1ra mRNA (icIL-1ra). Our results demonstrate the regulated presence of the icIL-1ra in the human endometrium. This finding supports a possible autocrine-paracrine role for the IL-1 system in the human endometrium and embryonic implantation.
TL;DR: It is suggested that LIF plays a regulatory role in trophoblast growth and differentiation during pregnancy in human placenta.
Abstract: Leukaemia inhibitory factor (LIF) is a cytokine that displays multiple activities in various tissues and is essential for blastocyst implantation in mice. In the human uterus, LIF is expressed in endometrial tissue and the decidua. To elucidate the role it plays, the mRNA levels for two LIF receptor (R) subunits, LIF-R and gp130, were examined in human endometrium, placenta and decidua by Northern blot hybridization. The expression of LIF-R gene was detected in the chorionic villus during the first trimester, in term placenta, and at lower levels in the decidua. The expression of LIF-R gene was not detectable in non-pregnant endometrium. The expression of the gp130 gene was detected in all tissues examined. During pregnancy, there was no significant change in the mRNA concentration of LIF-R in the placenta, while that of gp130 increased after the second trimester. The human choriocarcinoma cell line, BeWo, was found to express LIF-R and gp130. LIF inhibited forskolin-induced human chorionic gonadotrophin (HCG)-beta production by BeWo in a dose-dependent manner, and it ameliorated forskolin-induced growth suppression. These findings suggest that LIF plays a regulatory role in trophoblast growth and differentiation during pregnancy in human placenta.
TL;DR: Recombinant FSH, as measured in the rat Sertoli cell assay, was more potent than the urinary preparations Metrodin, Metrodin-HP and IS 70/45 with half maximal stimulation occurring at 2.2 +/- 0.3 IU/I, while the pituitary preparation IRP 83/575 was without effect on oestradiol and progesterone production at the highest dose.
Abstract: In this study the in-vitro biopotency and glycoform distribution of human recombinant follicle stimulating hormone (FSH, Org 32489) has been assessed. The biopotency of recombinant FSH was studied using animal (rat Sertoli) and human (granulosa-lutein) cell models. Recombinant FSH, as measured in the rat Sertoli cell assay, was more potent than the urinary preparations Metrodin, Metrodin-HP and IS 70/45 with half maximal stimulation (ED50; mean +/- SEM, n > 3) occurring at 2.2 +/- 0.5 IU/I (recombinant FSH), 4.7 +/- 1.1 IU/I (Metrodin), 13.2 +/- 0.7 IU/I (Metrodin-HP) and 6.4 +/- 0.3 IU/I (IS 70/45); the pituitary preparation IRP 83/575 had an ED50 of 10.4 +/- 0.1 IU/I. Using human granulosa-lutein cells, cultured for up to 4 days in the absence of exogenous steroid precursors, recombinant FSH was either without effect (three out of five patients) or inhibited both oestradiol and progesterone secretion. FSH (83/575) was without effect on oestradiol with preparations from any of the patients but slightly stimulated (134 +/- 8%; mean +/- SEM, P < 0.05) progesterone production at the highest dose (80 IU/I). The distribution of FSH isoforms, assessed by polyclonal radioimmunoassay, following chromatofocusing over the ranges pH < 3.5 and pH 3.5-7.0 respectively was recombinant FSH, 12.4 and 87.6%; Metrodin, 19.8 and 80.2%; Metrodin-HP, 50.2 and 49.8%; IS 70/45, 15.0 and 85.0%; IS 83/575, 70.9 and 29.1%. All glycoforms were pI < 7.0 for the five preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The difference in the time of expression of OCT4 mRNA and OCT6 mRNA indicates that the two genes play differential roles in human embryogenesis, and supports the concept that genetic elements determining developmental events during embryogenesis are conserved in evolution.
Abstract: Utilizing a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay system, the time course of mRNA expression of two transcription regulators, OCT4 and OCT6, was assessed in individual preimplantation human embryos. Examination of ova with three pronuclei and 1-cell through blastocyst stage embryos revealed that OCT4 mRNA was continuously expressed between the time of fertilization and 10+ cell stages, whereas OCT6 mRNA expression was not observed until the 10+ cell stage. The difference in the time of expression of OCT4 mRNA and OCT6 mRNA indicates that the two genes play differential roles in human embryogenesis. Nucleotide sequence homology for OCT4 and OCT6 among mammalian organisms supports the concept that genetic elements determining developmental events during embryogenesis are conserved in evolution.
TL;DR: It is demonstrated that in-vitro adhesion of first trimester human trophoblast to purified extracellular matrix proteins and to purified decidual stromal cell monolayers can be inhibited by monoclonal antibodies directed against appropriate integrin subunits and by synthetic peptides containing an arginine-glycine-aspartic acid sequence.
Abstract: At the time of implantation, the extracellular matrix proteins laminin and fibronectin are abundant in the decidua and are distributed pericellularly around each individual stromal cell. First trimester human trophoblast expresses both laminin and fibronectin receptors, specifically the alpha 1 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha 6 beta 4 integrin heterodimers. In this study we have demonstrated that in-vitro adhesion of first trimester human trophoblast to purified extracellular matrix proteins and to purified decidual stromal cell monolayers can be inhibited by monoclonal antibodies directed against appropriate integrin subunits and by synthetic peptides containing an arginine-glycine-aspartic acid sequence. Monoclonal antibodies (mAbs) to the alpha 5 and beta 1 integrin subunits and a synthetic peptide significantly inhibited adhesion to fibronectin. Binding of trophoblast to laminin was blocked with mAbs to the alpha 6 and beta 1 but not alpha 1 and beta 4 integrin subunits. Similarly, integrin-mediated adhesion to monolayers of decidual stromal cells could be blocked with mAbs to the alpha 5, alpha 6, beta 1 and beta 4 integrin subunits. Integrin-mediated signal transduction in normal and malignant trophoblast was investigated by Western blotting. A 115 kDa protein was the major tyrosine phosphorylated protein detected in trophoblast after binding to laminin or fibronectin. The profile of tyrosine phosphorylated proteins differed for malignant trophoblast.
TL;DR: Flow cytometry analysis was used for the accurate and objective evaluation of sperm chromatin condensation and chromatin stability of sperm nuclei and the influence of incubation on sperm Chromatin.
Abstract: Flow cytometry analysis was used for the accurate and objective evaluation of sperm chromatin condensation and chromatin stability of sperm nuclei. It was also possible to determine the influence of incubation on sperm chromatin. Different types of spermatozoa were studied: unprocessed spermatozoa at 1 and 45 min after ejaculation, after swim-up (migrated), spermatozoa incubated for 6 h in non-capacitating conditions (aged), or in B2 medium (capacitated) or B2 medium followed 1 h later with A23187 (reacted). All types of spermatozoa were analysed before and after treatment with various decondensation agents: sodium dodecyl sulphate (SDS), SDS plus EDTA and SDS plus disulphide-reducing agent [dithiotreitol (DTT)]. Sperm nuclei were enzymatically isolated and stained with propidium iodide. Three flow cytometric parameters were then measured: forward light scatter (cellular size), side light scatter (cellular complexity) and fluorescence (uptake of propidium iodide). Fluorescence was the most suitable parameter to study the degree of condensation and resistance to decondensation of DNA in the spermatozoa. Unprocessed spermatozoa 1 min after ejaculation underwent decondensation by all assessed treatments (anionic detergent, chelating or disulphide-reducing agents). Unprocessed spermatozoa 45 min after ejaculation and migrated spermatozoa did not undergo decondensation with SDS treatment, but decondensation occurred after treatment with SDS+EDTA or SDS+DTT. Spermatozoa incubated for 6 h under both non-capacitating (aged spermatozoa) and capacitating conditions (capacitated spermatozoa) and reacted spermatozoa were decondensed only after treatment with SDS+DTT.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Results indicate that, of the epidermal growth factors examined in this study, TGF alpha is locally synthesized in normal cycling human ovaries and TGFalpha may be synthesization in theca cells and act on the granulosa cells in a paracrine fashion through the EGFR in ovarian follicles.
Abstract: Immunolocalization of transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), cripto-1, amphiregulin and epidermal growth factor receptor (EGFR) was studied in 51 premenopausal human ovaries at various phases of the menstrual cycle. Localization of mRNA for TGF alpha and EGF was also studied by in-situ hybridization. Immunoreactive TGF alpha was observed predominantly in theca cells in 12 of 33 antral follicles in the follicular phase (6/14 dominant follicles, and 6/19 non-dominant) but not in any of the 18 follicles in the luteal phase or in primordial and pre-antral follicles. TGF alpha immunoreactivity was present predominantly in the luteinized granulosa cells in 13 of 15 corpora lutea in the luteal phase, which are considered to be active in steroidogenesis, but not in any of the regressed corpora lutea. Accumulation of TGF alpha mRNA hybridization signal was observed only in the theca cells in the follicles and luteinized theca cells in the ovaries that were immunohistochemically positive for TGF alpha. EGFR immunoreactivity was detected in 24 of 33 antral follicles in the follicular phase and in two of 18 follicles in the luteal phase but not in any of the corpora lutea. Immunoreactive EGF, cripto-1 and amphiregulin or EGF mRNA was not detected in any follicles, corpora lutea, or the stroma cells examined. These results indicate that, of the epidermal growth factors examined in this study, TGF alpha is locally synthesized in normal cycling human ovaries and TGF alpha may be synthesized in theca cells and act on the granulosa cells in a paracrine fashion through the EGFR in ovarian follicles.
TL;DR: It was determined that less than one nucleated erythrocyte per ml of maternal blood was of fetal origin, suggesting that the fetal cells are less suitable for fluorescence in-situ hybridization (FISH) analysis than similar preparations from other sources.
Abstract: The discovery of nucleated erythrocytes in maternal circulation provides a potential source for non-invasive prenatal diagnosis. We have evaluated the use of a three-stage procedure to determine the number of cells that are of fetal rather than maternal origin. First, monoclonal antibodies specific for CD45 and CD14 were used in conjunction with a magnetic (MACS) column to deplete unwanted leukocytes from maternal blood. This was followed by a positive MACS enrichment for nucleated erythrocytes, using an anti-CD71 (transferrin receptor) monoclonal antibody. To discriminate between fetal nucleated erythrocytes and those of maternal origin, enriched fractions were simultaneously stained with an anti-fetal haemoglobin (HbF) antibody and hybridized with probes specific for X and Y chromosomes. Samples were then subjected to blind analysis along with negative control samples from non-pregnant volunteers. Using this dual analysis, we were able to determine that less than one nucleated erythrocyte per ml of maternal blood was of fetal origin. Small numbers of these fetal cells were found in 87.5% of pregnancies, ranging from 6 to 35 weeks gestational age. Comparison of HbF and X/Y probe data also suggests that the fetal cells are less suitable for fluorescence in-situ hybridization (FISH) analysis than similar preparations from other sources.
TL;DR: It is suggested that cytosolic binding proteins modulate the supply of retinoic acid to the nuclei of endometrial cells during the menstrual cycle and that retinooic acid is involved in the cyclical control ofendometrial differentiation.
Abstract: Human endometrium is a glandular epithelial tissue with a substantial underlying stroma. Under the influence of ovarian steroids, endometrium undergoes a cyclical pattern of proliferation followed by secretory differentiation. Since retinoids promote the differentiation of many epithelia to secretory phenotypes they may be involved in controlling the secretory differentiation of human endometrial epithelium. Cytosolic binding proteins for retinol (cellular retinol binding protein) and retinoic acid (cellular retinoic acid binding protein) may play an important part in regulating the availability of retinoic acid to its nuclear receptors and we have therefore asked whether expression of mRNA for these proteins varies in relation to endometrial differentiation. In a series of 54 endometrial biopsies, both endometrial epithelial and stromal cells expressed mRNA for cellular retinol binding protein type I at a constant level throughout the menstrual cycle. Cellular retinoic acid binding protein type II was also expressed but the level of expression varied dramatically, being elevated in the proliferative phase and depressed during the secretory phase of the menstrual cycle in both epithelial and stromal cells. These data suggest that cytosolic binding proteins modulate the supply of retinoic acid to the nuclei of endometrial cells during the menstrual cycle and that retinoic acid is involved in the cyclical control of endometrial differentiation.
TL;DR: The composition of individual zonae pellucidae and modifications to this extracellular coat both before and after fertilization were analysed using a rapid, sensitive, non-radioactive biotinylation- or lectin-based detection system.
Abstract: The composition of individual human zonae pellucidae and modifications to this extracellular coat both before and after fertilization were analysed using a rapid, sensitive, non-radioactive biotinylation- or lectin-based detection system; these assays use commercially available reagents and can be performed on fragments of individual zonae pellucidae. The zona pellucida from unfertilized eggs is composed of three glycoprotein species designated as huZP1, huZP2 and huZP3. Under non-reducing conditions, the molecular weights of these proteins are approximately 150 kDa, approximately 100 kDa, and approximately 55-65 kDa respectively. Following fertilization, huZP1 was not detected under either non-reducing or reducing conditions. In contrast, after fertilization huZP2 was detected under non-reducing conditions, but not under reducing conditions. The ability to detect pre- and post-fertilization changes in a single human zona pellucida is discussed in relation to its value in assessing deficiencies in clinical and laboratory protocols used for in-vitro fertilization.
TL;DR: A positive correlation between the presence of certain beta 1-integrin cell adhesion molecules and the fertilizing ability of human spermatozoa suggests that integrins may be putative determinants in egg-sperm recognition and interaction.
Abstract: The purpose of this study was to investigate firstly whether beta 1-integrin cell adhesion molecules are expressed by human spermatozoa, and secondly whether there is any relationship between the expression of beta 1-integrin cell adhesion molecules and the fertilizing ability of human spermatozoa in vitro. A total of 50 semen samples were examined. The samples were obtained from the male partners of couples undergoing in-vitro fertilization (IVF) for either unexplained, tubal or male factor infertility. A panel of six monoclonal antibodies against beta 1-integrin cell adhesion molecules and immunohistochemical techniques were used to identify the presence of these molecules on the spermatozoa. The percentage of spermatozoa showing strong immunolabelling with each monoclonal antibody was assessed in each sample. The relationship between these results and the aetiology of infertility and incidence of fertilization was examined. beta 1-Integrins, and primarily the ones with alpha 4-, alpha 5- and alpha 6-chains, were expressed by human spermatozoa. Compared with semen samples from unexplained or male factor infertility patients, samples from tubal infertility patients had a significantly higher (P < 0.05) percentage of spermatozoa expressing adhesion molecules. There was a positive correlation between the expression of alpha 4, alpha 5 and alpha 6 adhesion molecules and the fertilizing ability of spermatozoa. The positive correlation between the presence of certain beta 1-integrin cell adhesion molecules and the fertilizing ability of human spermatozoa suggests that integrins may be putative determinants in egg-sperm recognition and interaction.
TL;DR: It is concluded that urinary VEGF/creatinine ratios increase following HCG stimulation, and circulating and urinary VPD values increase following gonadotrophin stimulation, in parallel with the increased ovarian vascularity.
Abstract: A recently identified cytokine, vascular endothelial growth factor (VEGF, vascular permeability factor) has been implicated in ovarian hyperstimulation syndrome in women undergoing assisted reproduction. We postulate that circulating and urinary VEGF values increase following gonadotrophin stimulation, in parallel with the increased ovarian vascularity. A VEGF radioimmunoassay was developed using iodinated VEGF as tracer, a goat anti-VEGF serum as antiserum and recombinant human VEGF as standard. The specificity of the assay was confirmed by comparing the reverse phase high-performance liquid chromatography (HPLC) pattern of VEGF immunoactivity in urine and urine spiked with recombinant VEGF. Urine was concentrated 5-fold prior to measurement by the radioimmunoassay. VEGF:creatinine ratios in early morning urine samples were used to monitor daily urinary VEGF concentrations based on its high correlation (r = 0.77, P < 0.001) with VEGF concentrations in 24 h urine collections. No diurnal variation in VEGF:creatinine ratios was detected. VEGF:creatinine ratios were determined daily from nine women undergoing gonadotrophin-releasing hormone (GnRH) agonist/gonadotrophin treatment. In a further 16 women, early morning urine samples were collected in the peri-ovulatory period. A significant increase (P < 0.005, n = 25) was observed in VEGF:creatinine ratios following human chorionic gonadotrophin (HCG) administration. VEGF:creatinine ratios correlated poorly (r < 0.34) with plasma oestradiol, follicle number and size. It is concluded that urinary VEGF/creatinine ratios increase following HCG stimulation.
TL;DR: The results suggest that p53 is overexpressed not only in malignant tumour cells but in certain trophoblast cell populations of the human placenta as well.
Abstract: We investigated the expression of the tumour-suppressor and cell cycle control protein p53 in human first trimester and term placenta, three choriocarcinoma cell lines (Jeg-3, JAR, BeWo) and human choriocarcinoma. Using monoclonal antibodies against p53 (DO-7, Ab-6, DO-1, PAb 1801), paraffin-embedded sections of first trimester and full-term placentae, human choriocarcinoma and Jeg-3, JAR and BeWo, as well as cytospins, were evaluated immunohistochemically. In addition, Western blots were carried out with the same antibodies on choriocarcinoma cell lines. In placentae, a small number of villous and extravillous cytotrophoblast cells, as well as very few syncytiotrophoblast cells, stained intensively. Also, p53 was visualized in some nuclei of the placental basal plate, whereas stroma and endothelium were negative for p53. Jeg-3, JAR and BeWo also showed a positive nuclear reaction with all applied antibodies. In paraffin-embedded sections of human choriocarcinoma, staining was confined to the nuclei of malignant cells. The results suggest that p53 is overexpressed not only in malignant tumour cells but in certain trophoblast cell populations of the human placenta as well.
TL;DR: It is demonstrated that both allelic drop-out and preferential amplification occur in somatic cells and suggest these are separate phenomena, and some inaccuracy/misdiagnosis may be due to both preferential amplification as well as allelic dropped-out.
Abstract: Previously the diagnosis of sex and cystic fibrosis status has been studied on single cells using the polymerase chain reaction (PCR). It has been suggested that allelic drop-out (PCR failure of one allele) and/or preferential amplification (hypo-amplification of one allele) may contribute to poor reliability and misdiagnosis, although this remains controversial as some reports suggest that allelic drop-out does not occur. We investigated an improved method of diagnosing sex and cystic fibrosis in single cells using a new technology (fluorescent PCR) to determine the base level of PCR artefacts (allelic drop-out and preferential amplification) which, in combination with improved sensitivity, should improve PCR reliability and accuracy. Fluorescent PCR gives high reliability (approximately 97%) and accuracy rates (approximately 97%) in somatic cells for both sex and cystic fibrosis diagnosis and its lower detection threshold allows allelic drop-out and preferential amplification to be easily distinguished. We also achieved high reliability and accuracy in diagnosing cystic fibrosis in human blastomeres. This study confirms earlier reports of both allelic drop-out and preferential amplification in single cell analysis. We demonstrate that both allelic drop-out and preferential amplification occur in somatic cells and suggest these are separate phenomena. Preferential amplification appeared common in single cell PCR while allelic drop-out apparently occurred at random in each allele. Preferential amplification was mainly amplification of the larger allele. We suggest that some inaccuracy/misdiagnosis may be due to both preferential amplification as well as allelic drop-out. Other findings were variability in drop-out between PCR and that amplification of signals from human blastomeres may be linked to embryo quality. We suggest that allelic drop-out is dependent on the number of cells within the sample.
TL;DR: The results suggest that SPMI is highly associated with the seminal coagulum components as very active forms that may adversely affect sperm motility when not properly processed after ejaculation.
Abstract: Human seminal plasma contains a sperm motility inhibitor (SPMI) originating from the seminal vesicles as a 52 kDa precursor form that is rapidly degraded by prostatic proteases after ejaculation. In this study, the distribution of SPMI biological activity and antigens was analysed in chemically induced, as well as naturally occurring, arrest of semen liquefaction. SPMI activity was detected exclusively in the coagulated semen fraction at 2200 +/- 560 IU, whereas total seminal plasma proteins separated more evenly between soluble and coagulated components (91 +/- 19 and 65 +/- 18 mg, respectively). An SPMI antiserum recognized different forms of SPMI precursors at 52, 38, 35, 33 and 20 kDa in the coagulum while the soluble protein fraction contained only one major immunoreactive band at 15 kDa. High levels of SPMI activity (1500 +/- 180 IU/ml) together with high molecular mass forms of SPMI precursor and low sperm motility (26%) were detected in semen samples that failed to liquefy spontaneously at room temperature. Addition of prostatic secretions to the non-liquefying samples caused a decrease of SPMI activity (330 +/- 17 IU/ml) and transformed the SPMI precursor into low molecular mass forms (14-22 kDa) with a concomitant increase in sperm motility to 49%. The results suggest that SPMI is highly associated with the seminal coagulum components as very active forms that may adversely affect sperm motility when not properly processed after ejaculation.
TL;DR: Findings indicate that abundant ADF is present in decidua and trophoblast cells; the localization of such a potent dithiol reducing substance may be beneficial in protecting the fertilized egg and placental trophoblasts from the cytotoxic effects of oxygen radicals.
Abstract: Adult T-cell leukaemia-derived factor (ADF), homologous to thioredoxin, displays various biological activities, such as radical scavenging action and the reduction of protein disulphide bonds. We examined the biochemical and immunohistochemical localization of ADF in the pregnant human uterus, using two heteroantibodies to ADF, antibody C and W. Immunohistochemically, decidua and trophoblast cells were intensely stained by antibody C. The concentration of ADF-like substance in the decidua was 95.9 ng/mg protein, determined by enzyme-linked immunosorbent assay. The molecular weight of ADF-like substance in these tissues was determined by gel electrophoresis to be 13 kDa, the same as that of recombinant ADF. These findings indicate that abundant ADF is present in decidua and trophoblast cells; the localization of such a potent dithiol reducing substance may be beneficial in protecting the fertilized egg and placental trophoblasts from the cytotoxic effects of oxygen radicals.
TL;DR: In this article, the presence of prepro-endothelin-I (ET-1) and the known receptor subtypes (ETA and ETB) in human endometrium at different stages of the menstrual cycle obtained at hysterectomy was investigated.
Abstract: This study was undertaken to investigate the presence of messenger RNA (mRNA) for prepro-endothelin-I (ET-1) and the known receptor subtypes (ETA and ETB) in human endometrium at different stages of the menstrual cycle obtained at hysterectomy. Northern blot analysis revealed expression of ET-1 mRNA in human endometrium during the normal menstrual cycle. The concentration of ET-1 mRNA in endometrial tissue was greater during the menstrual and proliferative phases than during the ovulatory and secretory phases. Immunoreactive ET-1 was secreted into the medium of isolated endometrial stromal cells. Oestradiol and progesterone significantly attenuated ET-1 release in endometrial stromal cells cultured for 6 days. ETA and ETB mRNA were also present in endometrial tissue of the normal cycle. The concentration of ETA receptor mRNA was greater in the proliferative phase than in the secretory phase, whereas expression of ETB mRNA increased in menstrual phase. ET-1 significantly increased extracellular accumulation of cyclic AMP (cAMP), intracellular generation of inositol phosphates and significantly enhanced DNA synthesis in cultured endometrial stromal cells from the proliferative phase. Our results showed that human endometrial cells synthesized and released ET-1, and contained ETA and ETB receptors which were functionally coupled to phosphoinositide breakdown and to adenylate cyclase with the increase of cAMP by ET-1 stimulation. Our findings suggest that ET-1 may have a potential autocrine and/or paracrine function in human endometrial stromal cells.