Pelvic organ prolapse (POP) and preterm premature rupture of the membranes (PPROM), two conditions which have in common weakening of the tensile strength of tissues, are thought to be caused, in part, by abnormal extracellular matrix synthesis and/or catabolism. We identified a new single nucleotide polymorphism (SNP) (NT_010194(LOXL1):g.45008784A>C) in the promoter of the LOXL1 gene, which is essential for elastin synthesis. Promoter studies showed that the minor “C” allele had significantly greater activity than the major “A” allele. Case-control studies examined the association of the alleles of this SNP with POP and PPROM. When comparing allele frequencies and genotypes in POP cases versus controls, no significant associations were found. A case-control study conducted in African-American neonates also found no significant associations between the promoter alleles and PPROM. We conclude that a functional SNP exists in the promoter region of LOXL1. Association studies suggest that the promoter SNP does not contribute significantly to risk of POP or PPROM.
Lysyl oxidase-like 1; pelvic organ prolapse; preterm premature rupture of membranes; elastin
To identify candidate genes contributing to preterm birth, we examined the existing literature on the association between known disorders of connective tissue synthesis and metabolism and related diseases and prematurity. Our hypothesis was that abnormal matrix metabolism contributes to prematurity by increasing risk of preterm premature rupture of membranes (PPROM) and cervical incompetence. Based on this review, we identified gene mutations inherited by the fetus that could predispose to preterm birth as a result PPROM. The responsible genes include COL5A1, COL5A2, COL3A1, COL1A1, COL1A2, TNXB, PLOD1, ADAMTS2, CRTAP, LEPRE1 and ZMPSTE24. Marfan syndrome, caused by FBN1 mutations, and polymorphisms in the COL1A1 and TGFB1 genes have been associated with cervical incompetence. We speculate that an analysis of sequence variation at the loci noted above will reveal polymorphisms that may contribute to susceptibility to PPROM in the general population.
Connective tissue; Genes; Preterm Birth; PPROM; Cervical incompetence
Ethnic disparity in preterm delivery between African Americans and European Americans has existed for decades, and is likely the consequence of multiple factors, including socioeconomic status, access to care, environment, and genetics. This review summarizes existing information on genetic variation and its association with preterm birth in African Americans. Candidate gene-based association studies, in which investigators have evaluated particular genes selected primarily because of their potential roles in the process of normal and pathological parturition, provide evidence that genetic contributions from both mother and fetus account for some of the disparity in preterm births. To date, most attention has been focused on genetic variation in pro- and anti-inflammatory cytokine genes and their respective receptors. These genes, particularly the pro-inflammatory cytokine genes and their receptors, are linked to matrix metabolism since these cytokines increase expression of matrix degrading metalloproteinases. However, the role that genetic variants that are different between populations play in preterm birth cannot yet be quantified. Future studies based on genome wide association or admixture mapping may reveal other genes that contribute to disparity in prematurity.
Genes; racial/ethnic disparities; preterm birth
Sex hormone-binding globulin (SHBG) is the primary plasma transport protein for sex steroid hormones and regulates the bioavailability of these hormones to target tissues. The gene encoding SHBG is complex and any of several polymorphisms in SHBG have been associated with alterations in circulating SHBG levels.
Epidemiological studies have revealed that low plasma SHBG levels are an early indicator of insulin resistance and predict the development of type 2 diabetes mellitus (T2DM) in both men and women. Although associations between low SHBG levels and risk of diabetes could be explained by the observation that elevations in insulin suppress hepatic SHBG production, recent studies documenting that the transmission of SHBG-altering polymorphisms are associated with risk of T2DM suggest that SHBG may have a more direct physiologic role in glucose homeostasis. However, the exact mechanism(s) underlying this association is not known.
Non-diabetic women with the polycystic ovary syndrome (PCOS), a common endocrine disorder that is associated with insulin resistance, similarly demonstrate lower levels of SHBG. In light of studies investigating polymorphisms in SHBG and T2DM, our group and others have hypothesized that SHBG may represent a candidate gene for PCOS. In this manuscript, we review studies investigating the association between SHBG polymorphisms and PCOS. In summary, multiple studies in women with PCOS confirm that certain genetic polymorphisms are associated with circulating SHBG levels, but they are not consistently associated with PCOS per se.
genetic polymorphisms; polycystic ovary syndrome; sex hormone binding globulin; type 2 diabetes mellitus
Preeclampsia is characterized by increased thromboxane and decreased prostacyclin levels, which predate symptoms, and can explain some of the clinical manifestations of preeclampsia, including hypertension and thrombosis. In this study, we examined DNA methylation of the promoter region of the thromboxane synthase gene (TBXAS1) and the expression of thromboxane synthase in systemic blood vessels of normal pregnant and preeclamptic women. Thromboxane synthase is responsible for the synthesis of thromboxane A2, a potent vasoconstrictor and activator of platelets. We also examined the effect of experimentally induced DNA hypomethylation on the expression of thromboxane synthase in a neutrophil-like cell line (HL-60 cells), and in cultured vascular smooth muscle and endothelial cells. We found that DNA methylation of the TBXAS1 promoter was decreased, and thromboxane synthase expression was increased in omental arteries of preeclamptic women as compared to normal pregnant women. Increased thromboxane synthase expression was observed in vascular smooth muscles cells, endothelial cells and infiltrating neutrophils. Experimentally induced DNA hypomethylation only increased expression of thromboxane synthase in the neutrophil-like cell line, whereas tumor necrosis factor-α, a neutrophil product, increased its expression in cultured vascular smooth muscle cells. Our study suggests that epigenetic mechanisms and release of tumor necrosis factor-α by infiltrating neutrophils could contribute to the increased expression of thromboxane synthase in maternal systemic blood vessels, contributing to the hypertension and coagulation abnormalities associated with preeclampsia.
preeclampsia; DNA methylation; thromboxane synthase; epigenetics; omental blood vessels; thromboxane A2
To determine if coexistence of periodontal disease (PD) and bacterial vaginosis (BV) is synergistic on the risk of spontaneous preterm delivery (sPTD).
Secondary analysis of a prospective cohort study. Women were screened 6–20 weeks gestation for PD and BV. Groups were defined by presence of BV and stratified on PD. The primary outcome was sPTD <37 weeks gestation. Univariable, stratified and multivariable analyses were performed to estimate the main and interaction effects of BV and PD on sPTD.
Of 1453 women screened, 792 (54.5%) were diagnosed with BV. Neither women with BV in the first trimester nor PD were at higher risk of sPTD (risk ratio (RR) for BV 1.1, 95% confidence interval (CI) 0.8–1.5, RR for PD 0.9, 95% CI 0.7–1.3). The interaction between BV and PD did not statistically significantly impact the odds of sPTD.
Coexistence of PD and BV did not have a synergistic effect on sPTD.
bacterial vaginosis; periodontal disease; preterm delivery
Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects 5–8% of reproductive age women. The primary features of PCOS are hyperandrogenemia, chronic anovulation and infertility. It has been suggested that defects in ovarian steroid metabolism contribute to the follicular growth arrest and abnormal production of ovarian steroid hormones that are characteristic of PCOS. 2-methoxyestradiol (2-ME) is formed by the action of catechol-O-methyltransferase (COMT) on 2-hydroxyestradiol. COMT expression is increased in the follicles and ovarian stroma of women with PCOS. Moreover, 2-ME decreases granulosa cell proliferation and steroidogenesis, raising the possibility that ovarian dysfunction associated with PCOS is due, in part, to increased synthesis of 2-ME resulting from increased COMT activity. Four single-nucleotide polymorphisms (SNPs) (rs6269, rs4633, rs4818, rs4680) in the COMT gene characterize haplotypes, which are associated with large variations in COMT enzymatic activity. The aim of this study was to determine whether individual COMT SNPs and the COMT haplotypes are associated with PCOS using a family-based test of association and linkage. Additionally, we examined the relationships between COMT SNPs and haplotypes with quantitative variables usually assessed in the evaluation of women with PCOS. There were no significant correlations between genotype and total testosterone, non-SHBG bound testosterone and BMI. However, we found that the prolactin level in women with PCOS varied significantly with COMT haplotype, and suggest that this association reflects a genetic factor influencing the stress response. Our findings suggest that common variants and haplotypes of the COMT gene are not major contributors to risk for PCOS, but that COMT genotype may influence prolactin levels.
Catechol-O-Methyltransferase; COMT; polycystic ovary syndrome; prolactin5
Polycystic ovary syndrome (PCOS) is a complex endocrine disorder with a strong familial component. PCOS is characterized by hyperandrogenemia and irregular menses. A recent genome wide association study of PCOS in a Chinese cohort identified three reproducible PCOS susceptibility loci mapping to 2p16.3 (luteinizing hormone/choriogonadotropin receptor; LHCGR), 2p21 (thyroid associated protein; THADA), and 9q33.3 (DENN/MADD domain containing 1A; DENNDIA). The impact of these loci in non-Chinese PCOS cohorts remains to be determined.
We tested association with PCOS of seven single nucleotide polymorphisms mapping to the three Chinese PCOS loci in two European-derived PCOS cohorts (Cohort A = 939 cases and 957 controls; Cohort B = 535 cases and 845 controls). Cases fulfilled the NICHD criteria for PCOS. Variation in DENND1A was strongly associated with PCOS in our cohort (pcombined cohorts=10−8 ); multiple variants in THADA were also associated with PCOS, while there was no significant evidence for association of LHCGR variation with PCOS. We had greater than 80% power to detect an effect of similar size as was observed by Chen et al. for DENND1A and THADA but reduced power (at <40%) for LHCGR at p=0.0001. We had sufficient power (57-88%) for LHCGR at p=0.01.
At least two of the PCOS susceptibility loci identified in the Chinese PCOS GWAS (DENND1A and THADA) are also associated with PCOS in European-derived populations, and therefore likely to be important in the etiology of PCOS regardless of ethnicity. Our analysis of the LHCGR gene was not sufficiently powered to detect modest effects.
PCOS; DENND1A; THADA; genome-wide association study
Sex hormone-binding globulin (SHBG) has emerged as one of the multiple genetic and environmental factors that potentially contribute to the pathophysiology of Type 2 diabetes mellitus (T2DM). In addition to epidemiologic studies demonstrating a consistent relationship between decreased levels of serum SHBG and incident T2DM, recent genetic studies also reveal that transmission of specific polymorphisms in the SHBG gene influence risk of T2DM. On the molecular level, elucidation of the multiple interactions between SHBG and its receptors in various target tissues, suggest physiologic roles for SHBG that are more complex than the simple transport of sex hormones in serum. Taken together, these data provide support for an expanded role of SHBG in the pathophysiology of insulin resistance and T2DM.
genetic polymorphisms; sex hormone binding globulin; type 2 diabetes mellitus
Preeclampsia, a pregnancy-specific disorder, is the third leading cause of maternal morbidity and mortality worldwide.
Method of Approach
To develop a device to detect preeclampsia in pregnant women living in low resource environments, a method was needed that had to be very low cost and, preferably, easily monitored by the woman herself. Due to the high cost and expertise involved in monitoring the two diagnostic criteria of preeclampsia (elevated blood pressure and proteinuria), edema, an indicator of preeclampsia was chosen instead.
The general principle of the method is to have each pregnant woman, early in pregnancy, fitted, on either her wrist or ankle, with a detection band, which is set to a preset expansion limit (e.g., expansion by 5%). When edema causes that body part to swell to the limit, the pregnant mother knows that she should seek medical assistance.
The resulting prototype device and calibration method requires little knowledge, and is very durable, cost-effective and portable.
preeclampsia indicator; edema detector
The study objectives were to evaluate adequacy of prenatal care and risk for preterm birth among Medicaid clients in Virginia and to determine if payment method is associated with the risk of preterm birth.
Birth certificate data for the Commonwealth of Virginia for 2007 and 2008 were linked with Medicaid claims data. Analysis was limited to singleton births. Three payment methods were evaluated: private insurance, self-pay, and Medicaid. The prevalence of preterm birth for each level of prenatal care defined by the Kotelchuck prenatal care index was assessed for each payment method. Unconditional logistic regression modeling was used to assess the association between payment method and preterm birth risk while controlling for known preterm birth risk factors.
Preterm birth prevalences (95% confidence interval [CI]) for the different payment methods were 7.9% (4.79-8.07) for the privately insured, 10.1% (9.57-10.60) for the self-pay group, and 10.2% (9.95-10.45) for Medicaid recipients. Compared with those with private insurance, women on Medicaid had an adjusted odds ratio (OR) for preterm birth (95% CI) of 0.99 (0.94-1.03). Self-pay mothers had a 32% increase in the odds of preterm birth relative to the privately insured. All payment groups show a trend toward significant reduction in preterm birth prevalence as adequacy of prenatal care improved from inadequate to adequate. Medicaid enrollees had a high prevalence of known risk factors, including smoking and illicit drug use and cervical insufficiency.
When known risk factors have been controlled, preterm birth risk for Medicaid enrollees did not differ significantly from the privately insured.
1) To determine allele frequencies of 3 LAMC1 single nucleotide polymorphisms (SNPs) in Caucasian and African-American (AA) women with > Stage II POP (cases) and in ethnicity-matched controls with < Stage 2 POP. 2) To determine if LAMC1 is associated with POP within ethnic groups.
Allelic discrimination was performed for LAMC1 SNPs rs10911193 (C/T), rs20563 (A/G), and rs20558 (T/C). SNP and haplotype-specific tests were used to examine associations between POP, ethnicity, and LAMC1.
411 women were enrolled. Significant differences in allele and haplotype frequencies existed amongst AA and Caucasians: rs10911193 “T” (p=0.0014); rs20563 “G” (p<0.0001); rs20558 “C” (p<0.0001); rs20563, rs20558 “GC” (p<0.0001); and rs20563, rs20558 “AT” (p<0.0001). No significant associations between POP and LAMC1 SNPs or haplotypes were found within ethnicities.
While significant differences were identified between AA and Caucasian women, no associations were found between any LAMC1 gene variant and advanced POP.
familial pelvic organ prolapsed; genetics; genetic predisposition; lamimin gamma 1; LAMC1; POP
The Spag16L gene codes for a protein that is localized to the central apparatus which is essential for normal sperm motility and male fertility. Sperm from mice homozygous for a targeted deletion of the Spag16L gene were examined to assess their flagellar motor functions compared with age- and strain-matched control sperm. Sperm were also demembranated with Triton X-100 and examined for their ability to respond to free calcium, as well as for their ability to undergo microtubule sliding driven by dynein action. In addition, the passive flagella, inhibited by sodium metavanadate to disable the dyneins, were examined for mechanical abnormalities. Live Spag16L-null sperm exhibited much less bending of the flagellum during the beat. The amount of microtubule sliding in the R-bend direction of the beat was selectively restricted, which suggests that there is limited activation of the dyneins on one side of the axoneme in the live cells. This is corroborated by the results on detergent-extracted sperm models. The flagellar response to calcium is greatly reduced. The calcium response requires the activation of the dyneins on outer doublets 1, 2, 3, and 4. These are the same dyneins required for R-bend formation. In axonemes prepared to disintegrate by microtubule sliding, we observed little or no extrusion of doublets 1 and 2, consistent with a reduced activity of their dyneins. This deficit in motor function, and an increased rigidity of the midpiece region which we detected in the passive flagella, together can explain the observed motility characteristics of the Spag16L-null sperm.
Mouse sperm lacking SPAG16L have altered motility and a reduced response to calcium.
axoneme; calcium; central pair; ciliopathies; dynein; hyperactivation
To determine if (1) birth outcomes among women on Medicaid differ significantly from outcomes of those with private insurance, after controlling for known risk factors, and (2) enhanced prenatal care influences care use and birth outcomes.
This is a review of studies published between 1989 and 2009 that examined birth outcomes (1) between women on Medicaid and those with private insurance and (2) among Medicaid enrollees who received comprehensive prenatal care.
When corrected for risk variables, birth outcomes are not different between private insurance and Medicaid patients. The impact of comprehensive prenatal care programs on birth outcomes varies across states and regions.
There is a need for critical evaluation of comprehensive programs in a regional and state context to determine opportunities for improvement.
Preeclampsia is a leading cause of perinatal morbidity and mortality. This disorder is thought to be multifactorial in origin, with multiple genes, environmental and social factors, contributing to disease. One proposed mechanism is placental hypoxia-driven imbalances in angiogenic and anti-angiogenic factors, causing endothelial cell dysfunction. Catechol-O-methyltransferase (Comt)-deficient pregnant mice have a preeclampsia phenotype that is reversed by exogenous 2-methoxyestradiol (2-ME), an estrogen metabolite generated by COMT. 2-ME inhibits Hypoxia Inducible Factor 1α, a transcription factor mediating hypoxic responses. COMT has been shown to interact with methylenetetrahydrofolate reductase (MTHFR), which modulates the availability of S-adenosylmethionine (SAM), a COMT cofactor. Variations in MTHFR have been associated with preeclampsia. By accounting for allelic variation in both genes, the role of COMT has been clarified. COMT allelic variation is linked to enzyme activity and four single nucleotide polymorphisms (SNPs) (rs6269, rs4633, rs4680, and rs4818) form haplotypes that characterize COMT activity. We tested for association between COMT haplotypes and the MTHFR 677 C→T polymorphism and preeclampsia risk in 1103 Chilean maternal-fetal dyads. The maternal ACCG COMT haplotype was associated with reduced risk for preeclampsia (P = 0.004), and that risk increased linearly from low to high activity haplotypes (P = 0.003). In fetal samples, we found that the fetal ATCA COMT haplotype and the fetal MTHFR minor “T” allele interact to increase preeclampsia risk (p = 0.022). We found a higher than expected number of patients with preeclampsia with both the fetal risk alleles alone (P = 0.052) and the fetal risk alleles in combination with a maternal balancing allele (P<0.001). This non-random distribution was not observed in controls (P = 0.341 and P = 0.219, respectively). Our findings demonstrate a role for both maternal and fetal COMT in preeclampsia and highlight the importance of including allelic variation in MTHFR.
Preterm birth is more prevalent in African Americans than European Americans and contributes to 3.4 times more African American infant deaths. Models of social inequity do not appreciably account for this marked disparity and molecular genetic studies have yet to characterize whether allelic differences that exist between races contribute to this gap. In this study, biometrical genetic models are applied to a large mixed-race sample consisting of 733,339 births to measure the extent that heritable factors and environmental exposures predict the timing of birth and explain differences between racial groups. Although we expected significant differences in mean gestational age between racial groups, we did not anticipate the variance of gestational age in African Americans (σ2 = 7.097) to be nearly twice that of European Americans (σ2 = 3.764). Our results show that this difference in the variance of gestational age can largely be attributed to environmental sources; which were 3.1 times greater in African Americans. Specifically, environmental factors that change between pregnancies, versus exposures that influence all pregnancies within a family, are largely responsible for the increased reproductive heterogeneity observed in African American mothers. Although the contribution of both fetal and maternal genetic factors differed between race categories, genetic studies may best be directed to understanding the differences in the socio-cultural sources of this heterogeneity, and their possible interaction with genetic differences within and between races. This study provides a comprehensive description of the relative genetic and environmental contributions to racial differences in gestational age.
The analysis of genetic and environmental contributions to preterm birth is not straightforward in family studies, as etiology could involve both maternal and fetal genes. Markov Chain Monte Carlo (MCMC) methods are presented as a flexible approach for defining user-specified covariance structures to handle multiple random effects and hierarchical dependencies inherent in children of twin (COT) studies of pregnancy outcomes. The proposed method is easily modified to allow for the study of gestational age as a continuous trait and as a binary outcome reflecting the presence or absence of preterm birth. Estimation of fetal and maternal genetic factors and the effect of the environment are demonstrated using MCMC methods implemented in WinBUGS and maximum likelihood methods in a Virginia COT sample comprising 7,061 births. In summary, although the contribution of maternal and fetal genetic factors was supported using both outcomes, additional births and/or extended relationships are required to precisely estimate both genetic effects simultaneously. We anticipate the flexibility of MCMC methods to handle increasingly complex models to be of particular relevance for the study of birth outcomes.
preterm birth; fetal; maternal; genetic; environment; MCMC; ML
Polycystic ovary syndrome (PCOS) is characterized by excessive theca cell androgen secretion, dependent upon LH, which acts through the intermediacy of 3′,5′-cyclic adenosine monophosphate (cAMP). cAMP signaling pathways are controlled through regulation of its synthesis by adenylyl cyclases, and cAMP degradation by phosphodiesterases (PDEs). PDE8A, a high-affinity cAMP-specific PDE is expressed in the ovary and testis. Leydig cells from mice with a targeted mutation in the Pde8a gene are sensitized to the action of LH in terms of testosterone production. These observations led us to evaluate the human PDE8A gene as a PCOS candidate gene, and the hypothesis that reduced PDE8A activity or expression would contribute to excessive ovarian androgen production. We identified a rare variant (R136Q; NM_002605.2 c.407G > A) and studied another known single nucleotide polymorphism (SNP) (rs62019510, N401S) in the PDE8A coding sequence causing non-synonymous amino acid substitutions, and a new SNP in the promoter region (NT_010274.16:g.490155G > A). Although PDE8A kinetics were consistent with reduced activity in theca cell lysates, study of the expressed variants did not confirm reduced activity in cell-free assays. Sub-cellular localization of the enzyme was also not different among the coding sequence variants. The PDE8A promoter SNP and a previously described promoter SNP did not affect promoter activity in in vitro assays. The more common coding sequence SNP (N401S), and the promoter SNPs were not associated with PCOS in our transmission/disequilibrium test-based analysis, nor where they associated with total testosterone or dehydroepiandrosterone sulfate levels. These findings exclude a significant role for PDE8A as a PCOS candidate gene, and as a Las major determinant of androgen levels in women.
PDE8A; polycystic ovary syndrome; androgens; theca; SNP
SOX5 is a transcription factor with homology to the high mobility group box region of the testis-determining factor, SRY. Both the mouse and human SOX5 genes encode a 48-kDa SOX5 protein (S-SOX5) that is only present in tissues containing cells with motile cilia/flagella. The mammalian sperm-associated antigen 6 gene (SPAG6) encodes an axoneme central apparatus protein. Because human and mouse SPAG6 gene promoters contain multiple potential binding sites for SOX5, SPAG6 gene regulation by S-SOX5 was investigated in BEAS-2B cells, a line derived from human bronchial cells. Like FOXJ1, a transcription factor known to be essential for motile ciliogenesis, S-SOX5 stimulated mouse and human SPAG6 promoter function in BEAS-2B cells, but the effect was abrogated when the SOX5 binding sites were mutated or deleted. S-SOX5 and FOXJ1 functioned cooperatively in stimulating SPAG6 promoter activity. The SPAG6 message was up-regulated when S-SOX5 was overexpressed in BEAS-2B cells, and silencing of S-SOX5 by RNA interference down-regulated SPAG6 transcripts. Chromatin immunoprecipitation and EMSA experiments demonstrated that S-SOX5 associates with the SPAG6 promoter directly. The present study demonstrates that SPAG6 is a S-SOX5 target gene, indicating a key role for S-SOX5 in the formation and function of motile cilia.
DNA-Protein Interaction; Gene Regulation; Promoters; Site-directed Mutagenesis; Sperm; Central Apparatus; Motile Cilia; SOX5; SPAG6; Transcription Regulation
The purpose of the study was to examine racial/ethnic differences in cervical insufficiency risk.
We used the US 2005 Natality data file. Analysis was limited to singleton births. The prevalence of cervical insufficiency was examined by the maternal characteristic for each racial group. Unconditional logistic regression modeling was used to assess the association between race and cervical insufficiency while controlling for confounders.
Cervical insufficiency risk for Black women was more than twice that for their White counterparts [odds ratio (OR) (95% confidence interval (CI)) of 2.45 (2.22–2.71)]. Prior pregnancy termination showed a dose–response relationship with cervical insufficiency. Compared with women with no history of prior pregnancy termination, primiparous women who have had one pregnancy termination had an OR (95% CI) of 2.49 (2.23–2.77). The OR for two, three and four or more terminations were 4.66 (4.07–5.33), 8.07 (6.77–9.61) and 12.36 (10.19–15.00), respectively. Other predictors of cervical insufficiency included previous preterm birth, parity, marital status, renal disease, history of diabetes, polyhydramnios and anemia.
There were significant racial/ethnic disparities with Black women having increased cervical insufficiency risk, independent of other studied factors. Prior pregnancy termination is also a major risk factor for cervical insufficiency. The White/Black disparity is evident in both primiparous and multiparous women.
cervical insufficiency; pregnancy termination; race; ethnicity
Worldwide, bacterial vaginosis (BV) is the most common vaginal disorder. It is associated with risk for preterm birth and HIV infection. The etiology of the condition has been debated for nearly half a century and the lack of knowledge about its cause and progression has stymied efforts to improve therapy and prevention. Gardnerella vaginalis was originally identified as the causative agent, but subsequent findings that it is commonly isolated from seemingly healthy women cast doubt on this claim. Recent studies shedding light on the virulence properties of G. vaginalis, however, have drawn the species back into the spotlight.
In this study, we sequenced the genomes of a strain of G. vaginalis from a healthy woman, and one from a woman with bacterial vaginosis. Comparative analysis of the genomes revealed significant divergence and in vitro studies indicated disparities in the virulence potential of the two strains. The commensal isolate exhibited reduced cytotoxicity and yet the cytolysin proteins encoded by the two strains were nearly identical, differing at a single amino acid, and were transcribed at similar levels. The BV-associated strain encoded a different variant of a biofilm associated protein gene and demonstrated greater adherence, aggregation, and biofilm formation. Using filters with different pore sizes, we found that direct contact between the bacteria and epithelial cells is required for cytotoxicity.
The results indicated that contact is required for cytotoxicity and suggested that reduced cytotoxicity in the commensal isolate could be due to impaired adherence. This study outlines two distinct genotypic variants of G. vaginalis, one apparently commensal and one pathogenic, and presents evidence for disparate virulence potentials.
Cells that morphologically and functionally resemble male germ cells can be spontaneously derived from ES cells. However, this process is inefficient and unpredictable suggesting that the expression pattern of male germ cell associated genes during spontaneous ES cell differentiation does not mimic the in vivo profiles of the genes. Thus, in the present study, the temporal expression profile of genes expressed at different stages of male germ cell development was examined in differentiating ES cells. The effect of all-trans retinoic acid (RA) which is a known inducer of primordial germ cell (PGC) proliferation/survival in vitro and testosterone which is required for spermatogenesis in vivo on the expression of these genes was also determined. Each of the 12 genes analyzed exhibited one of four temporal expression patterns in untreated differentiating ES cells: progressively decreased (Dpp3a, Sycp3, Msy2), initially low and then increased (Stra8, Sycp1, Dazl, Act, Prm1), initially decreased and then increased (Piwil2, Tex14), or relatively unchanged (Akap3, Odf2). RA-treated cells exhibited increased expression of Stra8, Dazl, Act, and Prm1 and suppressed expression of Dpp3a compared to untreated controls. Furthermore, testosterone increased expression of Stra8 while the combination of RA and testosterone synergistically increased expression of Act. Our findings establish a comprehensive profile of male germ cell gene expression during spontaneous differentiation of murine ES cells and describe the capacity of RA and testosterone to modulate the expression of these genes. Furthermore, these data represent an important first step in designing a plausible directed differentiation protocol for male germ cells.
Spermatogenesis; Gene Expression Profiling; Real-Time PCR; Embryoid Bodies
Mammalian SPAG16L, the orthologue of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites and the protein was confirmed to be phosphorylated in vivo. A yeast-two-hybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by co-immunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their co-localization was also noted by confocal microscopy in CHO cells where they were co-expressed. TSSK2 associates with SPAG16L via its C terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.
SPAG16L; kinase; phosphorylation; sperm motility
The study of germ cell-specific gene regulation in vitro is challenging. Here we report that the promoter of the oocyte-specific gene, Gdf9, is active in a population of cultured murine embryonic stem cells (ES) which have a phenotype resembling ooocytes. The promoter region of the murine Gdf9 coupled to enhanced green fluorescent protein (eGFP) was stably transfected into XX mouse ES cells. eGFP was expressed only in oocytes of chimeric mice generated from the transfected XX ES cells. The transfected ES cells were examined when cultured on feeder layers or as embryoid bodies. Large eGFP-positive cells, surrounded by a structure resembling a zona pellucida appeared transiently in cultures of the ES cells on feeder layers. Surprisingly, they were detectable on days 1 and 2 of culture but virtually absent on day 3. Addition of leukemia inhibitory factor (LIF) to the media significantly increased the number of eGFP-positive cels resembling oocytes. Quantitative real-time PCR demonstrated a parallel increase in Gdf9 and Zp3 mRNA with changes in the abundance of eGFP-positive cells. In embryoid body cultures, eGFP-positive cells appeared transiently and then re-appeared in regional clusters after 30−45 days of culture. These findings demonstrate that a population of cultured murine ES cells contain the transcriptional machinery to drive expression of an oocyte-specific gene, and that those cells phenotypically resemble oocytes.
Embryonic Stem cells; Gdf9; oocyte; leukemia inhibitory factor
This article traces the career of Celso-Ramon Garcia (1922–2004), noted physician, educator, and internationally renowned pioneer in the field of reproductive endocrinology. His work helped to formulate oral contraceptives used by millions of women throughout the world. Garcia's research collaborators included Gregory Pincus and John Rock, who together finalized the landmark clinical data needed to secure initial FDA approval for "the pill" in 1960. In addition to Garcia's monumental work in contraceptive endocrinology, his scholarly interests encompassed physiology of the menopause, minimally invasive reproductive surgery, as well as psychological aspects of infertility. Closely identified with the University of Pennsylvania, Garcia was instrumental in establishing the first formal clinical program in reproductive biology and influenced countless young scientists whose training he supervised and mentored. His distinguished career was emblematic of the best of the medical profession, characterized by compassion, intellect, and a sincere desire to help others. Our manuscript outlines Garcia's wide range of interests, acknowledges his superior fund of knowledge, and honors his humanitarian spirit – all of which contributed to an impressive legacy of medical discoveries. The impact of Prof. Garcia's work will continue to be felt for many years.