Association between endometrial thickness in oocyte donation cycles and pregnancy success rates.

Endometrial receptivity is a primary concern for embryo implantation success in fertility treatments. The present study was a retrospective analysis of 4070 cycles with donor oocytes and hormone-replacement therapy. Endometrial thickness was assessed once with transvaginal ultrasound. Patients were allowed to continue when endometrial thickness was ?5mm and had triple line morphology. Pregnancy rates, the number of gestational sacs and miscarriage rates were analysed in relation to endometrium status. Regression models were used to analyse associations, taking the day of embryo transfer into account. All patient parameters were homogeneous. Mean endometrial thickness was 7.24±1.66mm, the mean number of embryos transferred was 2.04±0.43, the pregnancy rate was 48.06% and sacs were present in 42.3% of cycles. There were no significant differences in pregnancy rates, number of gestational sacs and miscarriage rates for different endometrial thickness measurements. The present study is, to our knowledge, the largest study evaluating the role of endometrial thickness in oocyte donation cycles. Endometrial thickness >5mm is a reasonable parameter for determining treatment success, and once it is observed in a single ultrasonographic evaluation there is no need for subsequent monitoring and embryo transfer can be scheduled over the following 1-16 days, because the results are not compromised. This may lead to a significant reduction in time and cost in fertility clinics.

A 24-chromosome FISH technique in preimplantation genetic diagnosis: validation of the method.

Embryo screening for aneuploidy (AS) is part of preimplantation genetic diagnostics (PGD) and is aimed at improving the efficiency of assisted reproduction. Currently, several technologies, including the well-established fluorescence in situ hybridization (FISH) technique, cover the screening of all chromosomes in a single cell. This study evaluates a novel 24-chromosome FISH technique protocol (FISH-24). A total of 337 embryos were analyzed using the traditional 9-chromosome FISH technique (FISH-9) while 251 embryos were evaluated using the new FISH-24 technique. Embryos deemed nontransferable on Day 3 were cultured in vitro to Day 5 of development, then fixed and reanalyzed according to the technique allocated to each treatment cycle (107 embryos analyzed by FISH-9 and 111 by FISH-24). The global error rate (discrepancy between Day 3 and Day 5 results for a single embryo) was 2.8% after FISH-9 and 3.6% after FISH-24, with a p value of 0.95. Thus, we have established and validated a 24-chromosome FISH-based single cell aneuploidy screening technique, showing that the error rate obtained for FISH-24 is independent of the number of chromosomes analyzed and equivalent to the error rate observed for FISH-9, as a useful tool for chromosome segregation studies and clinical use.

Non-meiotic chromosome instability in human immature oocytes.

Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25-45 years of age) and 24 IVF oocyte donors (18-33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes.

A study of meiotic segregation in a fertile human population following ovarian stimulation with recombinant FSH-LH.

OBJECTIVE: The aim of the study is to investigate the meiotic segregation in fresh eggs from anonymous egg donors and to analyze the baseline levels of aneuploidy in this population. RESULTS: The study includes the largest series of donor eggs so far studied: 203 eggs from donors aged between 20 and 31 years. No diagnosis was obtained in 10.8 % of cases (22/ 203). The biopsy of the first and second polar bodies was completed in a sequential manner on day 0 and day 1 of embryo development. Chromosomes 13, 16, 18, 21 and 22 are analyzed by means of the FISH test. The diagnosable fertilized eggs gave an aneuploidy rate of 19.1 % (31/162), with 83.8 % (26/31) of the errors produced during meiosis I, 12.9 % (4/31) produced during meiosis II, and 3.2 % (1/31) produced during both meiosis I and II. The premature division of sister chromatids is the main source of meiotic error during Meiosis I, resulting in the creation of oocyte aneuploidy. CONCLUSIONS: FISH analysis of the first and second polar body in donor oocytes gave an aneuploidy rate of 19.1 %. This study shows the majority of errors occur during Meiosis I.

Comprehensive embryo analysis of advanced maternal age-related aneuploidies and mosaicism by short comparative genomic hybridization.

The short comparative genomic hybridization (short-CGH) method was used to perform a comprehensive cytogenetic study of isolated blastomeres from advanced maternal age embryos, discarded after fluorescent in situ hybridization (FISH) preimplantation genetic screening (PGS), detecting aneuploidies (38.5% of which corresponded to chromosomes not screened by 9-chromosome FISH), structural aberrations (31.8%), and mosaicism (77.3%). The short-CGH method was subsequently applied in one PGS, achieving a twin pregnancy.

Processing of semen can result in increased sperm DNA fragmentation.

Processing of semen for assisted reproductive technologies (ART) entails a number of procedures that include semen liquefaction, removal of seminal plasma by centrifugation, incubation, and cryopreservation. The results of this study indicate that incubation of semen at room temperature and semen cryopreservation can result in increased levels of sperm DNA fragmentation.

Mitochondrial organization in prepubertal goat oocytes during in vitro maturation and fertilization.

The aim of this study was to evaluate mitochondrial distribution during in vitro maturation (at 0, 15, 20, and 27 hr of IVM) and fertilization of prepubertal goat oocytes compared to mitochondrial distribution of ovulated and in vitro fertilized oocytes from adult goats. Oocytes from prepubertal goats were recovered from a slaughterhouse and were matured in M199 with hormones and serum for 27 hr. Ovulated oocytes were collected from gonadotrophin-treated Murciana goats. Frozen-thawed spermatozoa were selected by centrifugation in Percoll gradient and were capacitated in DMH with 20% steer serum for 1 hr. Ovulated and IVM-oocytes were inseminated in DMH medium with steer serum and calcium lactate for 20 hr. Oocytes and presumptive zygotes were stained with Mitotraker Green FM and observed under a confocal laser scanning microscope. Ultrastructural morphology of oocytes and presumptive zygotes were analyzed by transmission electron microscopy (TEM). Prepubertal goat oocytes at germinal vesicle stage (GV) presented mitochondria localized in the cortical and perinuclear region. IVM-oocytes at metaphase II presented mitochondria peripheral polarized to the region opposite were the metaphase spindle is positioned and within the polar body. Ovulated oocytes presented peripheral mitochondria distribution and mitochondrial aggregation around the MII spindle. At 20 hr post-insemination, mitochondria were distributed around the two synchronous pronuclei (2PN rpar; in zygotes ovulated oocytes whereas in prepubertal 2PN-zygotes mitochondria presented a peripheral polarized distribution. Images by TEM detected that immature prepubertal goat oocytes that are less electrodense and present fewer cristae than in vitro matured prepubertal goat oocytes; these are characterized by being associated to swollen vesicles. Mol. Reprod. Dev. 73: 617-626, 2006 (c) 2006 Wiley-Liss, Inc.

Self-correction of chromosomally abnormal embryos in culture and implications for stem cell production.

OBJECTIVE: To ascertain whether embryos classified by preimplantation genetic diagnosis (PGD) for infertility as abnormal and then plated to obtain stem cells would self-correct partially or totally in culture, producing disomic stem cells. DESIGN: Prospective study to determine the chromosome status of embryos on day 3 and 6, as well as cultured cells derived from inner cell masses from the same embryos when cultured up to day 12. SETTING: Research laboratory. PATIENT(S): Patients undergoing PGD of aneuploidy. INTERVENTION(S): Of 142 embryos classified by PGD for aneuploidy as abnormal, 50 were cultured to the blastocyst stage. At that stage a fraction of the embryos underwent trophectoderm biopsy to reconfirm the PGD diagnosis. After further co-culture with feeders up to day 12, 34 embryos attached to the feeder cells. Of those, 24 were analyzed by fluorescence in situ hybridization (FISH) and the rest for the expression of Oct-4, SSEA-3, SSEA-4, TRA1-60, and TRA1-80. MAIN OUTCOME MEASURE(S): Disomic cells obtained from trisomic embryos. RESULT(S): Analysis by FISH of day-12 cultures showed that 7 were totally normal, 6 were mostly abnormal, and 11 had experienced some chromosome normalization, having between 21% and 88% normal cells. Day-12 culture was positive for Oct-4 expression by reverse transcriptase polymerase chain reaction analysis and for SSEA-3, SSEA-4, TRA1-60, and TRA1-80 by immunocytochemistry. CONCLUSION(S): Chromosome self-normalization occurs in a significant proportion of chromosomally abnormal embryos, possibly because of the loss of a chromosome in trisomic cells after blastocyst stage. Thus chromosomally abnormal embryos are a potential source of disomic stem cells. Not all chromosomally abnormal embryos self-corrected. Abnormal stem cells that might be derived could be used as models to study the effect of chromosomal abnormalities on human development.

Microtubule and microfilament organization in immature, in vitro matured and in vitro fertilized prepubertal goat oocytes.

The aim of our study was to analyse the cytoskeletal organization of prepubertal goat oocytes. Microtubule and microfilament organization during in vitro maturation of prepubertal and adult goat oocytes and presumptive zygotes of in vitro matured-in vitro fertilized (IVM-IVF) prepubertal goat oocytes were analysed. Oocytes were matured in M-199 with hormones and serum and inseminated with frozen-thawed sermatozoa. Oocytes and presumptive zygotes were treated with anti-alpha-tubulin antibody and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody to stain the microtubules. Microfilaments were localized by means of phalloidin 5 microg/ml conjugated with fluorescein isothiocyanate (FITC-phalloidin). DNA was stained with propidium iodide. Stained oocytes were observed under a confocal laser scanning microscope. At the germinal vesicle nuclear stage, microfilaments were distributed at the cortex of the oocytes. After in vitro maturation, 91.7% of metaphase II (MII) oocytes from adult goats displayed microfilaments in the cortex and within the polar body and were characterized by the presence of a microfilament thickening at the cortical region over the meiotic spindle. In prepubertal goat MII oocytes only 5.7% of oocytes displayed microfilaments at the cortex and within the polar body. After insemination, most of the zygotes displayed microfilaments distributed at the cortex. An undefined microtubular network was observed in adult and prepubertal goat oocytes at the germinal vesicle stage. After in vitro maturation, 100% of MII oocytes from adult goats displayed microtubules on the meiotic spindle and within the polar body. This pattern of distribution was observed in 71.6% of prepubertal goat oocytes. Undefined microtubule networks were present in most of the zygotes analysed. In conclusion, cytoskeletal differences were found between prepubertal and adult goat MII oocytes. Furthermore, most of the zygotes from IVM-IVF prepubertal goat oocytes displayed cytoskeletal anomalies.

Differences in chromosome susceptibility to aneuploidy and survival to first trimester.

The purpose of this study was to find specific rates of aneuploidy in cleavage-stage embryos compared with first trimester data and to evaluate post-zygotic selection against aneuploidy. A total of 2058 embryos were analysed by flurorescence in-situ hybridization (FISH), and specific aneuploidy rates were obtained for 14 chromosomes. Data from morphologically abnormal embryos could be pooled with data from preimplantation genetic diagnosis (PGD) cycles because it was observed that they had similar rates of aneuploidy; thus, for the purpose of studying aneuploidy they could be, and were, pooled. Specific chromosome aneuploidy rates were not related to morphology or development of the embryos. The average maternal age of patients with aneuploid embryos was significantly higher than the overall analysed population. Monosomy appeared more commonly than trisomy. The chromosomes most frequently involved in aneuploidy were (in order) 22, 16, 21 and 15. When compared with first trimester pregnancy data, aneuploidies detected at cleavage stage seem to die in excess of 90% before reaching first trimester, with the exception of chromosome 16 and gonosomes (76% and 14% respectively). Differences in chromosome-specific aneuploidy rates at first trimester conceptions are probably produced by different chromosome-specific aneuploidy rates at cleavage stage and different survival rates to first trimester.

Improved implantation after preimplantation genetic diagnosis of aneuploidy.

The objective of this study was to assess the improvement in implantation rates after preimplantation genetic diagnosis (PGD) of numerical abnormalities for the sole indication of advanced maternal age when compared with a control group. Each PGD patient was matched to a control patient according to several parameters prior to obtaining pregnancy results. The diagnosis was based on the analysis of chromosomes X, Y, 13, 15, 16, 18, 21 and 22 plus a ninth probe (1, 7, 14 or 17) on a single cell per embryo. The results were also analysed in relation to the previous number of IVF cycles and the number of dipronucleated zygotes obtained, when replacing presumptively chromosomally normal embryos on day 4 of development. It was found that women of advanced reproductive age (average age 40 years) had a higher implantation rate (18%) than their matched controls treated with standard IVF (11%) (P < 0.05). This increase was not observed in patients with two or more previous IVF cycles or patients with fewer than eight zygotes. Patients with eight or more 2PN zygotes and one or no previous cycles showed the greatest improvement in implantation rate, from 8.8% in controls to 19.2% in the PGD group (average age 40 years) (P < 0.025).

Effect of Hoechst 33342 staining on developmental competence of prepubertal goat oocytes.

This study was undertaken to evaluate the effects of Hoechst staining on nuclear maturation and fertilisation when used at different stages of in vitro maturation (IVM) in prepubertal goat oocytes. Oocytes were matured in TCM1999 supplemented with 10% fetal bovine serum, 10 microg LH/ml, 10 microg FSH/ml and 1 microM 17beta-estradiol for 27 h. Frozen-thawed sperm cells were prepared by centrifugation in a discontinuous Percoll gradient and resuspended in DMH medium with 20% steer serum. Oocytes were fertilised in DMH medium with 7.75 mM calcium lactate. During IVM oocytes were exposed to 0.5 microg/ml of Hoechst 33342 staining and to ultraviolet light for a mean time of 3 s at 0 h, 8 h, 15 h, 20 h and 27 h. The percentage of metaphase II oocytes decreased significantly when oocytes were stained with Hoechst dye at 0 h, 8 h and 15 h of IVM. There was a decrease in total fertilisation rate and normal fertilisation rate of Hoechest-stained oocytes, independently of the time of Hoechst staining. Hoechst staining produces a significant reduction in oocyte viability when it is used in the early stages of in vitro maturation.

Blastomere fixation techniques and risk of misdiagnosis for preimplantation genetic diagnosis of aneuploidy.

One of the most critical steps in preimplantation genetic diagnosis (PGD) studies is the fixation required to obtain good fluorescence in-situ hybridization (FISH) nuclear quality without losing any of the cells analysed. Different fixation techniques have been described. The aim of this study was to compare three fixation methods (1, acetic acid/methanol; 2, Tween 20; 3, Tween 20 and acetic acid/methanol) based on number of cells lost after fixation, average rate of informative cells, rate of signal overlaps and FISH errors. A total of 100, 106 and 114 blastomeres were fixed using techniques 1, 2 and 3 respectively. Technique 2 gave the poorest nuclear quality with higher cytoplasm, number of overlaps and FISH errors. Although technique 1 showed better nuclear quality in terms of greater nuclear diameter, fewer overlaps and FISH errors, it is difficult to perform correctly. However, technique 3 shows reasonably good nuclear quality and is both easier to learn and use for PGD studies than the others.