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Category Archives: Human Reproduction

DNA integrity, growth pattern, spindle formation, chromosomal constitution and imprinting patterns of mouse oocytes from vitrified pre-antral follicles

BACKGROUND

Cryopreservation of follicles for culture and oocyte growth and maturation in vitro provides an option to increase the number of fertilizable oocytes and restore fertility in cases where transplantation of ovarian tissue poses a risk for malignant cell contamination. Vitrification for cryopreservation is fast and avoids ice crystal formation. However, the influences of exposure to high concentrations of cryoprotectants on follicle development, oocyte growth and maturation, and particularly, on the DNA integrity and methylation imprinting has not been studied systematically.

METHODS

Follicle survival and development, DNA damage, oocyte growth patterns, maturation, spindle formation and chromosomal constitution were studied after Cryo-Top vitrification of mouse pre-antral follicles cultured to the antral stage and induced to ovulate in vitro. Methylation of differentially methylated regions (DMRs) of two maternally (Snrpn and Igf2r) and one paternally (H19) imprinted genes was studied by bisulfite pyrosequencing.

RESULTS

Vitrification results in partial or total loss of oocyte–granulosa cell apposition and actin-rich transzonal projections, a transient increase in DNA breaks and a delay in follicle development. However, the oocyte growth pattern, maturation, spindle and chromosomal constitution are not significantly different between the vitrified and the control groups. Vitrification is not associated with elevated levels of imprinting mutations (aberrant methylation of the entire DMR), although the distribution of sporadic CpG methylation errors in the Snrpn DMR appears to differ slightly between control and vitrified oocytes.

CONCLUSIONS

DNA breaks appear to be rapidly repaired and vitrification of oocytes inside pre-antral follicles by the Cryo-Top method does not appear to increase risks of abnormal imprinting or disturbances in spindle formation and chromosome segregation.

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Effect of ICSI on gene expression and development of mouse preimplantation embryos

BACKGROUND

In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is however not known whether ICSI has additional effects on the transcriptome of mouse blastocysts.

METHODS

We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whitten's medium (ICSIWM) or KSOM medium with amino acids (ICSIKSOMaa) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo). In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip.

RESULTS

Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOMaa).

CONCLUSIONS

Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.

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Simulated physiological oocyte maturation (SPOM): a novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes

BACKGROUND

Oocyte in vitro maturation (IVM) reduces the need for gonadotrophin-induced ovarian hyperstimulation and its associated health risks but the unacceptably low conception/pregnancy rates have limited its clinical uptake. We report the development of a novel in vitro simulated physiological oocyte maturation (SPOM) system.

METHODS AND RESULTS

Bovine or mouse cumulus–oocyte complexes (COCs) were treated with cAMP modulators for the first 1–2 h in vitro (pre-IVM), increasing COC cAMP levels ~100-fold. To maintain oocyte cAMP levels and prevent precocious oocyte maturation, COCs were treated during IVM with an oocyte-specific phosphodiesterase inhibitor and simultaneously induced to mature with FSH. Using SPOM, the pre-IVM and IVM treatments synergized to increase bovine COC gap-junctional communication and slow meiotic progression (both P < 0.05 versus control), extending the normal IVM interval by 6 h in bovine and 4 h in mouse. FSH was required to complete maturation and this required epidermal growth factor signalling. These effects on COC had profound consequences for oocyte developmental potential. In serum-free conditions, SPOM increased bovine blastocyst yield (69 versus 27%) and improved blastocyst quality (184 versus 132 blastomeres; both P < 0.05 versus standard IVM). In mice, SPOM increased (all P < 0.05) blastocyst rate (86 versus 55%; SPOM versus control), implantation rate (53 versus 28%), fetal yield (26 versus 8%) and fetal weight (0.9 versus 0.5 g) to levels matching those of in vivo matured oocytes (conventional IVF).

CONCLUSIONS

SPOM is a new approach to IVM, mimicing some characteristics of oocyte maturation in vivo and substantially improving oocyte developmental outcomes. Adaption of SPOM for clinical application should have significant implications for infertility management and bring important benefits to patients.

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Testicular biopsy before ART: the patients’ perspective on the quality of care

BACKGROUND

So far, research on the patients’ perspective on fertility care has mainly focused on women. Our primary aim was to explore what is important to men with respect to care related to testicular sperm extraction (TESE) and to identify strengths and weaknesses of that care.

METHODS

This was a mixed-method study including phenomenology on interviews with 17 ‘interview participants’ (a purposive sample with diversification for the TESE result) who received a TESE treatment at a tertiary university clinic. Strengths and weaknesses of our TESE-related quality of care were identified. Additionally, a telephone questionnaire was answered by 15 ‘rating participants’ not willing to be interviewed and the questionnaire was analyzed quantitatively.

RESULTS

Interview participants wanted more than effective treatment and attached importance to the attitude of fertility clinic staff, information, time flow, personalized care, ‘all that is necessary’, coaching, a homely atmosphere, continuity, privacy and separate accommodation. The satisfaction of rating participants (independent of the TESE result) was problematic for ‘overall experience’, ‘physician at the day clinic’ and ‘gynecologist at discussion of the result’. The attitude of fertility clinic staff and information were the most obvious strengths of our TESE-related care. Weaknesses were lack of practical information on post-surgical recovery and waiting times in the waiting room.

CONCLUSIONS

TESE patients focus not only on clinical effectiveness but also on patient-centeredness of care, and this has led to organizational changes and a new patient information brochure in our center. Qualitative research is useful to examine, understand and improve the patient-centeredness of care.

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Comparison of IVF cycles reported in a voluntary ART registry with a mandatory registry in Spain

BACKGROUND

Monitoring assisted reproductive technology (ART) is essential to evaluate the performance of fertility treatment and its impact on birth rates. In Europe, there are two kinds of ART registers: voluntary and mandatory. The validity of register data is very important with respect to the quality of register-based observational studies. The aim of this paper is to determine the degree of agreement between voluntary and mandatory ART registers.

METHODS

The two sources for the data compared in this study (referring to 2005 and 2006) were FIVCAT.NET (an official compulsory Assisted Reproduction Registry within the Health Ministry of the Regional Government of Catalonia, to which all authorized clinics, both public and private, performing assisted reproduction in the region are obliged to report) and the register of the Spanish Fertility Society (SEF), to which data are provided on a voluntary basis. The SEF register data were divided into two groups: (i) data from clinics in Catalonia (SEF-CAT); (ii) data from the rest of Spain, excluding Catalonia (SEF-wCAT). The techniques compared were IVF cycle using patients’ own eggs (IVF cycle) versus donor egg cycles.

RESULTS

For IVF cycles, the voluntary ART register reflected 77.2% of those on the official one, but the corresponding figure was only 34.4% with respect to donated eggs. The variables analysed in the IVF cycle (insemination technique used, patients’ age, number of embryos transferred, pregnancy rates, multiple pregnancies and deliveries) were similar in the three groups studied. However, we observed significant differences in donor egg cycles with regard to the insemination technique used, pregnancy rates and multiple pregnancies between the voluntary and the official register.

CONCLUSIONS

Data from the voluntary ART register for IVF cycles are valid, but those for donor egg cycles are not. Further study is necessary to determine the reasons for this difference.

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Aberrant expression of regulators of cell-fate found in eutopic endometrium is found in matched ectopic endometrium among women and in a baboon model of endometriosis

BACKGROUND

We have recently shown that women with endometriosis express an increased amount of telomerase and nucleolin, with concomitant loss of -H2AX in eutopic endometrium. To further examine these selected factors that regulate cell fate, in the pathogenesis of endometriosis, we studied the expression of telomerase, nucleolin, proliferating cell nuclear antigen and -H2AX in ectopic endometriotic deposits from women, and in matched eutopic and ectopic endometrial tissue from a baboon model of endometriosis.

METHODS

Ectopic active peritoneal endometriotic lesions were collected from seven symptomatic women. Endometriosis was induced in six baboons by intra-peritoneal autologous inoculation of menstrual endometrium. Eutopic and matched ectopic endometrial tissues were collected prior to and 6, 12 and 15 months after the induction of endometriosis as previously described. Eutopic endometrium was also obtained from eight healthy fertile control baboons. Immunohistochemistry was performed as previously described, and telomerase activity was confirmed using the telomeric repeat amplification protocol assay.

RESULTS

All active human endometriotic lesions expressed the proliferative markers but showed weak or absent staining for -H2AX. A similar expression pattern of these markers was seen in the ectopic lesions of the baboons with induced disease. In these baboons, the eutopic endometrium also showed intense immunoreactivity for all proliferative markers 6–12 months after induction with a parallel loss of -H2AX. The opposite staining pattern was seen in eutopic endometrium of healthy animals and in pre-induction endometrium of animals with induced disease.

CONCLUSIONS

Endometriotic lesions have excess proliferative potential; in baboons, these were present within 12 months of the initiation of the disease. In eutopic tissue, these changes appear to be induced by the development of endometriosis.

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