To improve the biologic understanding of the Polycystic Ovarian Syndrome (PCOS) condition by examining the circadian variation and relationship between Anti Müllerian Hormone (AMH), gonadotropins and ovarian steroids in PCOS patients compared to normally ovulating and menstruating women. By comparing the pattern of co-variation between AMH and Luteinizing Hormone, two compounds closely linked to hyperandrogenism and anovulation in PCOS, the involvement of the Hypothalamic-Pituitary-Ovarian axis in PCOS pathology could be elucidated.
Eight normal-weighted young, anovulatory PCOS-women as study group and ten normal menstruating and ovulating women as controls.
Observational prospective study of the circadian variation in AMH, gonadotropins, sex steroids and androgens in a study and a control group. A circadian profile was performed in each study and control subject during a 24-h period by blood sampling every second hour, starting at 8:00 a.m. and continuing until 8:00 a.m. the following day.
Significant differences in hormonal levels were found between the groups, with higher concentrations of AMH, LH and androgens in the PCOS group and lower amounts of FSH and progesterone. A distinct difference in the circadian variation pattern of AMH and LH between PCOS patients and normal controls was seen, with PCOS patients presenting a uniform pattern in serum levels of AMH and LH throughout the study period, without significant nadir late-night values as was seen in the control group. In PCOS women, a significant positive association between LH/ FSH and testosterone was found opposite to controls.
Main outcome measures
Circadian variation in Anti-Müllerian Hormone, gonadotropins and ovarian steroids and the covariation between them.
A significant difference in the circadian secretion of LH and AMH in PCOS women compared to normally ovulating women indicate an increased GnRH pulse, creating high and constant LH serum concentrations. A significant co-variation between LH and AMH may suggest LH as a factor involved in the control of AMH secretion.
The measurement of ovarian volume has been shown to be a useful indirect indicator of the ovarian reserve in women of reproductive age, in the diagnosis and management of a number of disorders of puberty and adult reproductive function, and is under investigation as a screening tool for ovarian cancer. To date there is no normative model of ovarian volume throughout life. By searching the published literature for ovarian volume in healthy females, and using our own data from multiple sources (combined n = 59,994) we have generated and robustly validated the first model of ovarian volume from conception to 82 years of age. This model shows that 69% of the variation in ovarian volume is due to age alone. We have shown that in the average case ovarian volume rises from 0.7 mL (95% CI 0.4–1.1 mL) at 2 years of age to a peak of 7.7 mL (95% CI 6.5–9.2 mL) at 20 years of age with a subsequent decline to about 2.8 mL (95% CI 2.7–2.9 mL) at the menopause and smaller volumes thereafter. Our model allows us to generate normal values and ranges for ovarian volume throughout life. This is the first validated normative model of ovarian volume from conception to old age; it will be of use in the diagnosis and management of a number of diverse gynaecological and reproductive conditions in females from birth to menopause and beyond.
Numerous common genetic variants have been linked to blood pressure, but no underlying mechanism has been elucidated. Population studies have revealed that the variant rs5068 (A/G) in the 3′ untranslated region of NPPA, the gene encoding atrial natriuretic peptide (ANP), is associated with blood pressure. We selected individuals on the basis of rs5068 genotype (AG vs. AA) and fed them a low- or high-salt diet for 1 week, after which they were challenged with an intravenous saline infusion. On both diets, before and after saline administration, ANP levels were up to 50% higher in AG individuals than in AA individuals, a difference comparable to the changes induced by high-salt diet or saline infusion. In contrast, B-type natriuretic peptide levels did not differ by rs5068 genotype. We identified a microRNA, miR-425, that is expressed in human atria and ventricles and is predicted to bind the sequence spanning rs5068 for the A, but not the G, allele. miR-425 silenced NPPA mRNA in an allele-specific manner, with the G allele conferring resistance to miR-425. This study identifies miR-425 as a regulator of ANP production, raising the possibility that miR-425 antagonists could be used to treat disorders of salt overload, including hypertension and heart failure.
The need for practice guidelines for fertility preservation in young women with hematological malignancies has been increased. To develop recommendations, publications relevant to fertility preservation and hematological cancers were identified through a PubMed database search and reviewed systematically, focusing on the effects of oncological treatments on fertility as well as on the efficacy, feasibility and risks of existing fertility preservation methods.
Hematological malignancy; Fertility; Fertility preservation; Chemotherapy; Lymphoma; Leukemia, cancer
To assess the longevity of ovarian grafts in five cancer patients who underwent heterotopic autotransplantation of frozen-thawed ovarian tissue.
Five cancer survivors underwent heterotopic ovarian transplantation between 2001and 2011. Stored ovarian tissue (for 1–10 years) was rapidly thawed and transplanted into the space between the rectus sheath and the rectus muscle (8–20 cortical sections per patient). Endocrine function was assessed by monthly blood tests (FSH, LH, E2, progesterone and testosterone) and ultrasound after transplantation. The monitoring was continued until the cessation of endocrine function by consecutive blood tests (E2 < 20 pg/ml; FSH ≥ 35 IU/L).
Endocrine function was restored in all patients between 12–20 weeks after transplantation. Four patients required the second transplantation one to two years after the first transplantation. The duration of endocrine function after the second transplantation was much longer (9 months–84 months). The longest duration of endocrine function was seen in a woman who underwent ovarian transplantation in 2003 and 2004 after radiotherapy for cervical cancer. Even more than seven years after transplantation, endocrine function has not ceased (FSH 9.5, E2 108, on July 1, 2011). Of note, this patient underwent three IVF cycles in 2004 which resulted in four embryos.
Long-term endocrine function lasting for seven years can be established with heterotopic transplantation of cryobanked human ovarian tissue. Re-establishment of long-term endocrine function after ovarian transplantation will benefit young cancer survivors with premature ovarian failure.
Fertility preservation; Ovarian transplantation; Heterotopic transplantation; Endocrine function; Cancer; Cryopreservation; Ovarian tissue
When a young woman is diagnosed with breast cancer, there is often a sense of urgency by the patient and her providers to initiate treatment. This article provides guidelines for incorporating the discussion of fertility preservation with newly diagnosed young women with breast cancer.
Fertility preservation; Breast cancer; Chemotherapy; Cancer; Survivorship; Oocyte cryopreservation; Embryo cryopreservation; Ovarian tissue cryopreservation; Ovarian stimulation; Infertility
Because of its ethical and social implications, preimplantation sex selection is frequently the subject of debates.
In 2006, we surveyed specialists in reproductive medicine in Germany using an anonymous questionnaire, including sociodemographic data and questions regarding ethical problems occurring in the practice of reproductive medicine. Most questions focused on preimplantation sex selection, including 10 case vignettes, since these enabled us to describe the most difficult and ethically controversial situations. This is the first survey among specialists in reproductive medicine regarding this topic in Germany.
114 specialists in reproductive medicine participated, 72 males (63%) and 42 females (37%), average age was 48 years (age range 29–67 years). The majority of respondents (79%) favoured a regulation that limits the use of preimplantation sex selection only for medical reasons, such as X-linked diseases (including 18%: summoning an ethics commission for every case). A minority of 18% approved of the use of sex selection for non-medical reasons (4% generally and further 14% for family balancing). 90% had received obvious requests from patients. The highest approval (46%) got the counselling guideline against a preimplantation sex selection and advising a normal pregnancy, if preimplantation sex selection would be allowed in Germany. The majority (67%) was opposed the personal use of preimplantation sex selection for non-medical reasons, but would think about it in medical cases. In opposite to woman, 14% of the men were in favour of personal use for non-medical reasons (p = 0,043). 25% of specialists in reproductive medicine feared that an allowance of preimplantation sex selection would cause a shift in the sex ratio.
The majority of German specialists in reproductive medicine opposes preimplantation sex selection for non-medical reasons while recommending preimplantation sex selection for medical reasons, e.g. X-linked diseases like haemophilia.
The majority of ovarian primordial follicles must be preserved in a quiescent state to allow for the regular production of gametes over the female reproductive lifespan. However, the molecular mechanism that maintains the long quiescence of primordial follicles is poorly understood. Under certain pathological conditions, the entire pool of primordial follicles matures simultaneously leading to an accelerated loss of primordial follicles and to premature ovarian failure (POF). We have previously shown that loss of Pten (phosphatase and tensin homolog deleted on chromosome ten) in mouse oocytes leads to premature activation of the entire pool of primordial follicles, subsequent follicular depletion in early adulthood, and the onset of POF. Lack of PTEN leads to increased phosphatidylinositol 3-kinase (PI3K)–Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling in the oocytes. To study the functional and pathological roles of elevated mTORC1 signaling in the oocytes, we treated the Pten-mutant mice with the specific mTORC1 inhibitor rapamycin. When administered to Pten-deficient mice prior to the activation of the primordial follicles, rapamycin effectively prevented global follicular activation and preserved the ovarian reserve. These results provide a rationale for exploring the possible use of rapamycin as a drug for the preservation of the primordial follicle pool, and the possible prevention of POF.
Estrous cycle disruption after spinal cord injury (SCI) in female rats is a common phenomenon. It remains unknown, however, if the aberrant estrous cycle is a result of an injury to the spinal cord itself or due to the general stress associated with surgical interventions. We addressed this issue by determining estrous cyclicality in female rats after a spinal cord hemisection (HX), implantation of EMG wires into selected hindlimb muscles, and/or injections of tracer dyes into the spinal cord. Since it is known that aerobic exercise can enhance the recovery of locomotor function in rodents with an incomplete SCI, we also determined if locomotor training positively impacts the disrupted estrous cycle after a HX. Estrous cycle assessments were made during a 5-8 week period in 27 female rats before and after HX, EMG, and/or dye injection surgeries and in HX rats that recovered spontaneously or underwent locomotor training. Our results show that estrous cyclicality was disrupted (cycle length >5 days) in approximately 76%, 46%, and 50% of the rats after HX, EMG, and dye injection surgeries, respectively. The cyclicality, however, was disrupted for a longer period after HX than after EMG or dye injection surgeries. Furthermore, estrous cycle mean length was shorter in the trained than non-trained HX group. These results suggest that estrous cycle disruption after a major SCI is a consequence of both the direct injury to the spinal cord and to the associated stress. Moreover, moderate aerobic exercise initiated early after a spinal cord HX returns the duration of the estrous cycle towards normal.
spinal cord injury; surgical intervention; EMG surgery; estrous cycle; rodents
Background & Aims
Krüppel-like factor 5 (KLF5) is transcription factor that is expressed by dividing epithelial cells of the intestinal epithelium. KLF5 promotes proliferation in vitro and in vivo and is induced by mitogens and various stress stimuli. To study the role of KLF5 in intestinal epithelial homeostasis, we examined the phenotype of mice with conditional deletion of Klf5 in the gut.
Mice were generated with intestinal-specific deletion of Klf5 (Vil-Cre;Klf5fl/fl).
Morphological changes in the small intestine and colon were examined by immunohistochemistry, immunoblotting, and real-time PCR.
Klf5 mutant mice were born at a normal Mendelian ratio but had high mortality compared to controls. Complete deletion of Klf5 from the intestinal mucosa resulted in neonatal lethality that corresponded with an absence of epithelial proliferation. Variegated intestinal-specific deletion of Klf5 in adult mice resulted in morphological changes that included a regenerative phenotype, impaired barrier function, and inflammation. Adult mutant mice exhibited defects in epithelial differentiation and migration. These changes were associated with reduced expression of Cdx 1, Cdx2, and Eph and ephrin signaling proteins. Concomitantly, Wnt signaling to β-catenin was reduced. Proliferation in regenerative crypts was associated with increased expression of the progenitor cell marker Sox9.
Deletion of Klf5 in the gut epithelium of mice demonstrated that KLF5 maintains epithelial proliferation, differentiation, and cell positioning along the crypt radial axis. Morphological changes that occur with deletion of Klf5 are associated with disruption of canonical Wnt signaling and increased expression of Sox9.
intestinal homeostasis; gastrointestinal development; genetics; GI tract
Primordial ovarian follicles, which are often present in the ovaries of premature ovarian failure (POF) patients or are cryopreserved from the ovaries of young cancer patients who are undergoing gonadotoxic anticancer therapies, cannot be used to generate mature oocytes for in vitro fertilization (IVF). There has been very little success in triggering growth of primordial follicles to obtain fertilizable oocytes due to the poor understanding of the biology of primordial follicle activation.
We have recently reported that PTEN (phosphatase and tensin homolog deleted on chromosome ten) prevents primordial follicle activation in mice, and deletion of Pten from the oocytes of primordial follicles leads to follicular activation. Consequently, the PTEN inhibitor has been successfully used in vitro to activate primordial follicles in both mouse and human ovaries. These results suggest that PTEN inhibitors could be used in ovarian culture medium to trigger the activation of primordial follicle. To study the safety and efficacy of the use of such inhibitors, we activated primordial follicles from neonatal mouse ovaries by transient treatment with a PTEN inhibitor bpV(HOpic). These ovaries were then transplanted under the kidney capsules of recipient mice to generate mature oocytes. The mature oocytes were fertilized in vitro and progeny mice were obtained after embryo transfer.
Results and Conclusions
Long-term monitoring up to the second generation of progeny mice showed that the mice were reproductively active and were free from any overt signs or symptoms of chronic illnesses. Our results indicate that the use of PTEN inhibitors could be a safe and effective way of generating mature human oocytes for use in novel IVF techniques.
Fertility issues should be addressed to all patients in reproductive age before cancer treatment. In men, cryopreservation of sperm should be offered to all cancer patients in reproductive age regardless of the risk of gonadal failure. In women, the recommendation of fertility preservation should be individualized based on multiple factors such as the urgency of treatment, the age of the patient, the marital status, the regimen and dosage of cancer treatment.
Fertility preservation; Cancer; Lymphoma; Leukemia; Breast cancer; Chemotherapy; Ovarian tissue cryopreservation; Oocyte cryopreservation; Primary ovarian insufficiency; Cancer survival; Gonadal failure; GnRH agonist; Fertility
Ovarian cryopreservation is one option for fertility preservation in patients with cancer. The danger of reseeding malignancies could be eliminated by in vitro maturation of primordial follicles from the frozen-thawed tissue. However, the development of this system is hindered by uncertainties regarding factors that activate primordial follicles. Neuronal growth factors such as vasoactive intestinal peptide (VIP) play important roles in early mammalian folliculogenesis. There are no data on the expression of VIP and its vasoactive intestinal peptide pituitary adenylate cyclase 1 and 2 receptors (VPAC1-R and VPAC2-R) in human preantral follicles.
Tissue samples from 14 human fetal ovaries and 40 ovaries from girls/women were prepared to test for the expression of VIP, VPAC1-R, and VPAC2-R on the protein (immunohistochemisty) and mRNA (reverse transcription polymerase chain reaction) levels. Immunohistochemistry staining was mostly weak, especially in fetal samples. The VIP protein was identified in oocytes and granulosa cells (GCs) in the fetal samples from 22 gestational weeks (GW) onwards. In girls/women, VIP follicular staining (oocytes and GCs) was identified in 45% of samples. VPAC1-R protein was identified in follicles in all fetal samples from 22GW onwards and in 63% of the samples from girls/women (GC staining only in 40%). VPAC2-R protein was identified in follicles in 33% of fetal samples and 47% of the samples from girls/women. The mRNA transcripts for VIP, VPAC1-R, and VPAC2-R were identified in ovarian extracts from fetuses and women.
VIP and its two receptors are expressed in human ovarian preantral follicles. However, their weak staining suggests they have limited roles in early follicular growth. To elucidate if VIP activates human primordial follicles, it should be added to the culture medium.
BACKGROUND & AIMS
Krüppel-like factor 5 (KLF5) is a transcription factor that promotes proliferation; is highly expressed in dividing crypt cells of the gastrointestinal epithelium and is induced by various stress stimuli. We sought to determine the role of KLF5 in colonic inflammation and recovery by studying mice with dextran sulfate sodium (DSS)-induced colitis.
Wild-type (WT) and Klf5+/− mice were given DSS in the drinking water to induce colitis. For recovery experiments, mice were given normal drinking water for 5 days after DSS administration. The extent of colitis was determined using established clinical and histological scoring systems. Immunohistochemical and immunoblotting analyses were used to examine proliferation, migration, and expression of the epidermal growth factor receptor (EGFR).
Klf5 expression was increased in colonic tissues of WT mice given DSS; induction of Klf5 was downstream of mitogen-activated protein kinase signaling. In DSS-induced colitis, Klf5+/− mice exhibited greater sensitivity to DSS than WT mice, with significantly higher clinical and histological colitis scores. In recovery experiments, Klf5+/− mice showed poor recovery, with continued weight loss and higher mortality than WT mice. Klf5+/− mice from the recovery period had reduced epithelial proliferation and cell migration at sites of ulceration compared to WT mice; these reductions correlated with reduced expression of EGFR.
Epithelial repair is an important aspect of recovery from DSS-induced colitis.
The transcription factor KLF5 regulates mucosal healing through its effects on epithelial proliferation and migration.
inflammatory bowel disease; animal model; inflammatory response; mouse
Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism.
TGF-β blockade significantly slows tumor growth through many mechanisms, including activation of CD8+ T-cells and macrophages. Here, we show that TGF-β blockade also increases neutrophil-attracting chemokines resulting in an influx of CD11b+/Ly6G+ tumor-associated neutrophils (TAN) that are hypersegmented, more cytotoxic to tumor cells, and express higher levels of pro-inflammatory cytokines. Accordingly, following TGF-β blockade, depletion of these neutrophils significantly blunts anti-tumor effects of treatment and reduces CD8+ T-cell activation. In contrast, in control tumors, neutrophil depletion decreases tumor growth and results in more activated CD8+ T-cells intra-tumorally. Together, these data suggest that TGF-β within the tumor microenvironment induces a population of TAN with a pro-tumor phenotype. TGF-β blockade results in the recruitment and activation of TAN with an anti-tumor phenotype.
tumor immunology; immunosuppression; TGFβ; tumor associated macrophages; Tumor associated neutrophils; lung cancer; mesothelioma
Background Although human leukocyte antigen (HLA) DQ and
DR loci appear to confer the strongest genetic risk for
type 1 diabetes, more detailed information is required for other loci within the
HLA region to understand causality and stratify additional risk factors. The
Type 1 Diabetes Genetics Consortium (T1DGC) study design included
high-resolution genotyping of HLA-A, B,
C, DRB1, DQ, and
DP loci in all affected sibling pair and trio families, and
cases and controls, recruited from four networks worldwide, for analysis with
clinical phenotypes and immunological markers.
Purpose In this article, we present the operational strategy of training,
classification, reporting, and quality control of HLA genotyping in four
laboratories on three continents over nearly 5 years.
Methods Methods to standardize HLA genotyping at eight loci included: central
training and initial certification testing; the use of uniform reagents,
protocols, instrumentation, and software versions; an automated data transfer;
and the use of standardized nomenclature and allele databases. We implemented a
rigorous and consistent quality control process, reinforced by repeated
workshops, yearly meetings, and telephone conferences.
Results A total of 15,246 samples have been HLA genotyped at eight loci to
four-digit resolution; an additional 6797 samples have been HLA genotyped at two
loci. The genotyping repeat rate decreased significantly over time, with an
estimated unresolved Mendelian inconsistency rate of 0.21%. Annual
quality control exercises tested 2192 genotypes (4384 alleles) and achieved
99.82% intra-laboratory and 99.68% inter-laboratory
Limitations The chosen genotyping platform was unable to distinguish many allele
combinations, which would require further multiple stepwise testing to resolve.
For these combinations, a standard allele assignment was agreed upon, allowing
further analysis if required.
Conclusions High-resolution HLA genotyping can be performed in multiple laboratories
using standard equipment, reagents, protocols, software, and communication to
produce consistent and reproducible data with minimal systematic error. Many of
the strategies used in this study are generally applicable to other large
Locally-produced TGF-β promotes tumor-induced immunosuppression and contributes to resistance to immunotherapy. This paper explores the potential for increased efficacy when combining immunotherapies with TGF-β suppression using the TGF-β type I receptor kinase inhibitor, SM16. Adenovirus expressing IFNβ (Ad.IFNβ) was injected intratumorally once in established subcutaneous AB12 (mesothelioma) and LKR (lung cancer) tumors or intratracheally in a K-ras orthotopic lung tumor model. Mice bearing TC1 (lung cancer) tumors were vaccinated with two injections of adenovirus expressing HPV-E7 (Ad.E7). SM16 was administered orally in formulated chow. Tumor growth was assessed and cytokine-expression and cell populations were measured in tumors and spleens by real time-PCR and flow cytometry. SM16 potentiated the efficacy of both immunotherapies in each of the models and caused changes in the tumor microenvironment. The combination of SM16 and Ad.INFβ increased the number of intratumoral leukocytes (including macrophages, NK cells, and CD8+ cells) and increased the percentage of T-cells expressing the activation marker CD25. SM16 also augmented the anti-tumor effects of Ad.E7 in the TC1 flank tumor model. The combination did not increase HPV-E7 tetramer-positive CD8+ T cells in the spleens, but did induce a marked increase in the tumors. Tumors from SM16-treated mice showed increased mRNA and protein for immunostimulatory cytokines and chemokines, as well as endothelial adhesion molecules, suggesting a mechanism for the increased intratumoral leukocyte trafficking. Blockade of the TGF-β signaling pathway augments the anti-tumor effects of Ad.INFβ immune-activating or Ad.E7 vaccination therapy. The addition of TGF-β blocking agents in clinical trials of immunotherapies may increase efficacy.
tumor immunology; immunosuppression; TGFβ; tumor associated macrophages; cytokines; lung cancer; mesothelioma; tumor vaccine; interferon-β
During the last years, several studies have reported the significant relationship between the production of soluble HLA-G molecules (sHLA-G) by 48–72 hours early embryos and an increased implantation rate in IVF protocols. As consequence, the detection of HLA-G modulation was suggested as a marker to identify the best embryos to be transferred. On the opposite, no suitable markers are available for the oocyte selection.
The major finding of the present paper is that the release of ICAM-1 might be predictive of oocyte maturation. The results obtained are confirmed using three independent methodologies, such as ELISA, Bio-Plex assay and Western blotting. The sICAM-1 release is very high in immature oocytes, decrease in mature oocytes and become even lower in in vitro fertilized embryos. No significant differences were observed in the levels of sICAM-1 release between immature oocytes with different morphological characteristics. On the contrary, when the mature oocytes were subdivided accordingly to morphological criteria, the mean sICAM-I levels in grade 1 oocytes were significantly decreased when compared to grade 2 and 3 oocytes.
The reduction of the number of fertilized oocytes and transferred embryos represents the main target of assisted reproductive medicine. We propose sICAM-1 as a biochemical marker for oocyte maturation and grading, with a possible interesting rebound in assisted reproduction techniques.
Progress in oncology could improve survival rate in children, but would probably lead to impaired fertility and puberty. In pre-pubertal girls, the only therapeutic option is the cryopreservation of one ovary. Three births have been reported after reimplantation of cryopreserved mature ovary. Conversely, reimplantation of ovary preserved before puberty (defined as immature ovary) has never been performed in humans.
In order to analyze ovarian function, we performed transplantation using fresh or cryopreserved immature grafts in pre-pubertal or adult mice. Puberty as well as cyclic hormonal activity was restored. All follicle populations were present although a significant reduction in follicle density was observed with or without cryopreservation. Although fertility was restored, the graft is of limited life span. Because ex vivo ovary manipulation and cryopreservation procedure, the status of genomic imprinting was investigated. Methylation status of the H19 and Lit1 Imprinting Control Regions in kidney, muscle and tongue of offsprings from grafted mice does not show significant alteration when compared to those of unoperated mice.
These results demonstrate that immature ovarian grafting can restore spontaneous puberty and fertility. However, these data suggest that follicle depletion leads to premature ovarian failure. This study addresses the very important epigenetics issue, and provides valuable information to the study of ovarian transplantation suggesting that these procedures do not perturb normal epigenetics marks. These results are highly relevant to the reimplantation question of immature cortex in women.
Purpose: To investigate the difference of in vitro and in vivo grown oocytes, we compared maturation, fertilization, development, and maternal gene expression from both in vitro and in vivo grown mouse oocytes.
Methods: The preantral follicles isolated from 12-day-old mice were cultured on Transwell-COL membrane inserts. After culture, maturation, fertilization, and developmental rates were assessed. RT-PCR (reverse transcription—polymerase chain reaction) was performed to examine the expression of β-actin, GDF-9, and IGF-II in matured oocytes.
Results: No difference in the nuclear maturation was detected between in vitro and in vivo grown oocytes, but the mean oocyte diameter of the in vitro group was smaller than that of the in vivo group. The fertilization rate was significantly lower in the in vitro group than in the in vivo group (p < 0.05). The capacities of in vitro grown oocyte to cleave and develop to blastocysts were significantly lower than those of the in vivo grown oocytes (p < 0.001). Moreover, blastocyst of in vitro group had fewer total cells than those of in vivo group (p < 0.05). In regards to the expression of genes in mature oocytes, growth differentiation factor-9 (GDF-9) expression was similar between the two groups, but β-actin was significantly reduced in the in vitro group compared to the in vivo group. Particularly, the expression of insulin-like growth factor II (IGF-II) was not found in the in vitro grown oocytes.
Conclusions: These results showed that in vitro grown oocytes did not have the same developmental capacity as in vivo grown oocytes. We assume that the aberrant expression of maternal-derived genes in the in vitro grown oocytes may cause the poor embryo viability.
Gene expression; in vitro; maturation; mouse; preantral follicle
Micro computed tomography (micro CT) has been shown to provide exceptionally high quality imaging of the fine structural detail within urinary calculi. We tested the idea that micro CT might also be used to identify the mineral composition of urinary stones non-destructively.
Micro CT x-ray attenuation values were measured for mineral that was positively identified by infrared microspectroscopy (FT-IR). To do this, human urinary stones were sectioned with a diamond wire saw. The cut surface was explored by FT-IR and regions of pure mineral were evaluated by micro CT to correlate x-ray attenuation values with mineral content. Additionally, intact stones were imaged with micro CT to visualize internal morphology and map the distribution of specific mineral components in 3-D.
Micro CT images taken just beneath the cut surface of urinary stones showed excellent resolution of structural detail that could be correlated with structure visible in the optical image mode of FT-IR. Regions of pure mineral were not difficult to find by FT-IR for most stones and such regions could be localized on micro CT images of the cut surface. This was not true, however, for two brushite stones tested; in these, brushite was closely intermixed with calcium oxalate. Micro CT x-ray attenuation values were collected for six minerals that could be found in regions that appeared to be pure, including uric acid (3515 – 4995 micro CT attenuation units, AU), struvite (7242 – 7969 AU), cystine (8619 – 9921 AU), calcium oxalate dihydrate (13815 – 15797 AU), calcium oxalate monohydrate (16297 – 18449 AU), and hydroxyapatite (21144 – 23121 AU). These AU values did not overlap. Analysis of intact stones showed excellent resolution of structural detail and could discriminate multiple mineral types within heterogeneous stones.
Micro CT gives excellent structural detail of urinary stones, and these results demonstrate the feasibility of identifying and localizing most of the common mineral types found in urinary calculi using laboratory CT.