Comparison of two different oocyte vitrification methods: a prospective, paired study on the same genetic background and stimulation protocol.

STUDY QUESTION: Can two different methods for oocyte vitrification, one using an open tool and the other a closed tool, result in similar oocyte survival rates? SUMMARY ANSWER: The oocyte survival rate was found to be higher in the closed method. WHAT IS KNOWN ALREADY: Open vitrification is performed routinely in oocyte donation cycles. Closed oocyte vitrification may result in slower cooling rates and thus it is less used, even though it has been recommended in order to avoid the risk of cross-contamination between material from different patients. STUDY DESIGN, SIZE, DURATION: This is a prospective cohort study with sibling oocytes carried out in a fertility center between July 2014 and January 2016. The study included 83 oocyte donors each providing a minimum of 12 mature oocytes (metaphase II: MII) at oocyte retrieval. Oocyte survival rate and fertilization rate, as well as reproductive outcomes (biochemical, clinical, ongoing pregnancy and live birth rates) per embryo transfer and also cumulatively between the two methods were compared by Chi2 tests. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donor oocytes were denuded and six MII oocytes from each donor were vitrified using an open method and later assigned to one recipient, while another six MII oocytes were vitrified using a closed method and assigned to a different recipient (paired analysis). ICSI was used in all cases and embryo transfer was performed on Day 2-3 in all cases. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte donors were 24.8 years old on average (SD 4.7). Recipient age (average 41.2 years, SD 4.7) and BMI (mean 23.8 kg/m2, SD 4.0) were similar between recipient groups. Oocytes vitrified using the closed method had higher survival rate (94.5% versus 88.9%, P = 0.002), but lower fertilization rate (57.1% versus 69.8%, P < 0.001) compared to the open method. The number of fresh embryos transferred in the two groups was 1.8 on average (SD 0.4). Biochemical (45% closed versus 50% open), clinical (40% versus 50%) and ongoing (37.5% versus 42.5%) pregnancy rates were not different between groups (P > 0.05) and neither were live birth rates (37.5% versus 42.5%, P > 0.05). Cumulative reproductive results (obtained after the transfer of all the embryos) were also similar between groups. LIMITATIONS, REASONS FOR CAUTION: The participants of this study were oocyte donors, i.e. young women in good health, and care should be exerted in extending our results to other populations such as infertility patients, oncofertility patients and women freezing oocytes to delay childbearing. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that, in spite of different survival and fertilization rates, closed and open oocyte vitrification methods should offer similar reproductive outcomes up to cumulative live birth rates. STUDY FUNDING/COMPETING INTEREST(S): The authors report no conflict of interest. Vitrolife provided the media and the closed method tool needed for the study at no cost.

Is there a relation between the time to ICSI and the reproductive outcomes?

STUDY QUESTION: Does time to ICSI affect reproductive outcomes? SUMMARY ANSWER: Biochemical and clinical pregnancy diminish progressively as time between oocyte pick up (OPU) and ICSI increases after fresh embryo transfer. WHAT IS KNOWN ALREADY: Appropriate oocyte cytoplasmic and nuclear maturation are of paramount importance to ensure an optimal embryonic developmental competence. While nuclear maturation is usually attained by the time an oocyte reaches OPU, cytoplasmic maturation cannot be readily assessed and might be incomplete. On the other hand, excessive in vitro culture of mature human oocytes can affect their ultrastructural characteristics and, in mice, induces alterations in gene expression and changes of chromatin and histone modification patterns. STUDY DESIGN, SIZE, DURATION: Retrospective consecutive cohort study including 1468 ICSI cycles carried out in a single center between December 2012 and September 2015. All cycles were with patient's own oocytes and fresh embryo transfer (ET). A radiofrequency-based system was used to record exact culture times, namely, OPU-denudation (DN); DN-ICSI and OPU-ICSI. We analyzed the effect of total and partial time intervals between procedures, from OPU to ICSI, on fertilization rate and biochemical, clinical, ongoing pregnancy and live birth rates. PARTICIPANTS/MATERIALS, SETTING, METHODS: Differences in laboratory times between positive and negative biochemical, clinical, ongoing pregnancies and birth results were tested by Mann-Whitney U test. The likelihood of positive clinical outcomes was further modeled by locally weighted scatterplot smoothing (LOWESS) regression and logistic regression, adjusting for woman's age and BMI, number of transferred embryos; mean embryo morphological score, sperm origin and status, and number of mature oocytes obtained at OPU. Effect of time on fertilization rate was modeled by Generalized Linear Modeling (GLM) and LOWESS regression. MAIN RESULTS AND THE ROLE OF CHANCE: The mean woman's age was 38.4 years (SD 4.6). Biochemical, clinical, ongoing pregnancy and live birth rates after the fresh ET were: 39.6, 33.1, 25.7 and 20.8%, respectively. Cumulative values for biochemical pregnancy and live birth were 46.4 and 26.3%, respectively. Mean times in hours for OPU-DN, DN-ICSI and OPU-ICSI were: 1.00 (SD 0.20); 3.86 (SD 1.93) and 4.87 (SD 1.96), respectively, and were not different for pregnant and non-pregnant patients. However, multivariate analyses showed that on average (anti-log transformed), each 1-h increase in the OPU-ICSI time reduced the likelihood of biochemical pregnancy by 7.3% (95% CI: 0.7-13.5%) and of clinical pregnancy by 7.7% (95% CI 0.8-14.1%), after the fresh ET. No effect of time was observed for ongoing pregnancy or live birth rates. Increasing OPU-ICSI time increased the fertilization rate (B = 0.052, 95% CI: 0.022, 0.082). LIMITATIONS, REASONS FOR CAUTION: The lack of relationship between incubation time of oocytes and live birth rates might be due to uncontrolled variables. Given the population analyzed, these results should not be extended to other ART protocols such as in vitro maturation of oocytes or classical IVF fertilization. WIDER IMPLICATIONS OF THE FINDINGS: This study indicates that in vitro ageing of mature oocytes significantly affects the chances to become pregnant. Effect on live birth rates, although not evident in this study, cannot be excluded. Limiting incubation time of mature oocytes in the embryology laboratory should improve reproductive results for patients using their own oocytes and with a transfer of fresh embryos. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: NA.

Should we worry about the clock? Relationship between time to ICSI and reproductive outcomes in cycles with fresh and vitrified oocytes.

STUDY QUESTION: Is there an optimal time to perform ICSI with respect to the times of oocyte pick-up (OPU), in order to maximize the reproductive outcomes in cycles with fresh and vitrified/warmed donor oocytes? SUMMARY ANSWER: We found no significant differences in reproductive outcomes of ICSI cycles within a wide range of times between OPU and ICSI. WHAT IS KNOWN ALREADY: In assisted reproduction, the oocyte is subject to denudation, vitrification/warming and ICSI. As shorter interaction with cumulus cells, oocyte ageing in vitro and insufficient recovery after warming may all impact the resulting embryo developmental competence, strictly controlled times between procedures are often implemented. However, most protocols have not been tested with the aim to improve reproductive results, and little information is available on the ideal times to be followed during these steps in order to optimize fertilization rates and embryo quality, and to achieve the highest pregnancy rate. STUDY DESIGN, SIZE, DURATION: Data from 3986 ICSI cycles performed between December 2012 and May 2014 were included (3178 with fresh and 808 with vitrified/warmed donor oocytes). PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI was performed using donor oocytes and either partner or donor sperm. Exact times between OPU, denudation, vitrification, warming and ICSI were recorded automatically by a radiofrequency-based system. OPU was performed strictly 36 h after GnRH agonist trigger. Biochemical pregnancy was defined as a positive serum ßHCG 15 days after transfer, clinical pregnancy was defined as a visible embryo with heartbeat 5 weeks after transfer, and ongoing pregnancy was defined as a normally developing pregnancy at 12 weeks after transfer. MAIN RESULTS AND THE ROLE OF CHANCE: Times between OPU and ICSI (OPU-ICSI) ranged from 1 h 25 min to 17 h 13 min (averagefresh ± SD = 4 h 58 m ± 1 h; averagevitrified= 9 h 18 m ± 2 h). We found no effect of OPU-ICSI time on fertilization rate (pfresh=0.39; pvitrified=0.86) or embryo quality at Days 2 and 3 (pfresh=0.08; pvitrified=0.22). There was no difference in average OPU-ICSI times between positive and negative pregnancies (biochemical, clinical, ongoing and live birth rates) in either fresh (P = 0.71, 0.43, 0.79, 0.96) or vitrified (P = 0.59, 0.33, 0.73, 0.87) oocytes, respectively. Data were adjusted for oocyte donor age, semen status, number of motile spermatozoa and sperm concentration, and no effect of OPU-ICSI time on pregnancy and live birth rates for either fresh (P = 0.57, 0.16, 0.11, 0.46) or vitrified (P = 0.80, 0.73, 0.91, 0.95) oocytes was found. Further analysis for linear trend using OPU-ICSI time categorized in deciles showed that pregnancy rates and live birth rates do not increase or decrease across deciles. We found no effect of time taken for denudation to vitrification, warming to ICSI and denudation to ICSI on pregnancy rates. LIMITATIONS, REASONS FOR CAUTION: This is a study with automatically collected times from a high number of ICSI cases; however, its retrospective nature cannot exclude the influence of unaccounted for variables on the results. All oocytes came from oocyte donors (≤35 years old), so results cannot be extended to older or infertile women. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that the effective window of time for insemination by ICSI might be wider than previously thought. It therefore appears that, within appropriate time frames, the management of ICSI cycles involving oocytes from young women in embryology laboratories could be adjusted to accommodate caseloads and workflow with no loss of oocyte viability or cycle efficiency.

Paternal age and assisted reproductive outcomes in ICSI donor oocytes: is there an effect of older fathers?

STUDY QUESTION: Does paternal age affect semen quality and reproductive outcomes in oocyte donor cycles with ICSI? SUMMARY ANSWER: Paternal age is associated with a decrease in sperm quality, however it does not affect either pregnancy or live birth rates in reproductive treatments when the oocytes come from donors <36 years old and ICSI is used. WHAT IS KNOWN ALREADY: The weight of evidence suggest that paternal age is associated with decreasing sperm quality, but uncertainty remains as to whether reproductive outcomes are affected. Although developed to treat severe sperm factor infertility, ICSI is gaining popularity and is often used even in the presence of mild male factor infertility. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study spanning the period between February 2007 and June 2010. A total of 4887 oocyte donation cycles were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fertilization was carried out by ICSI in all cycles included, and the semen sample used was from the male partner in all cases. The association of male age with semen parameters (volume, concentration, percentage of motile spermatozoa) was analyzed by multiple analysis of covariance. The association of male age with reproductive outcomes (biochemical pregnancy, miscarriage, ongoing pregnancy and live birth rate) was modeled by logistic regression, where the following covariates were introduced: donor age, recipient age, semen state (fresh versus frozen) and number of transferred embryos (3 and 2 versus 1). MAIN RESULTS AND THE ROLE OF CHANCE: We identified a significant relationship between paternal age and all sperm parameters analyzed: for every 5 years of age, sperm volume decreases by 0.22 ml (P < 0.001), concentration increases by 3.1 million sperm/ml (P = 0.003) and percentage motile spermatozoa decreases by 1.2% (P < 0.001). No differences were found in reproductive outcomes (biochemical pregnancy, miscarriage, clinical pregnancy, ongoing pregnancy and live birth) among different male age groups. LIMITATIONS, REASONS FOR CAUTION: The use of donor oocytes, while extremely useful in highlighting the role of male age in reproductive outcomes, limits the generalization of our results to a population of young women with older male partners. No data were available on perinatal and obstetrical outcomes of these pregnancies. Most (75%) cycles used frozen/thawed sperm samples which might have introduced a bias owing to loss of viability after thawing. ICSI was performed in all cycles to control for fertilization method; this technique could mask the natural fertilization rate of poorer sperm samples. Furthermore, we did not use stringent ICSI indications; and our data are therefore not generalizable to cases where only severe male factor is considered. However, male patients were of different racial background, thus allowing generalizing our results to a wider patient base. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggests that paternal age does not affect reproductive outcomes when the oocyte donor is <36 years of age, indicating that ICSI and oocyte quality can jointly overcome the lower reproductive potential of older semen. STUDY FUNDING/COMPETING INTERESTS: This study was supported in part by Fundació Privada EUGIN. The authors have no conflicts of interest to declare.

First successful double-factor PGD for Lynch syndrome: monogenic analysis and comprehensive aneuploidy screening.

Preimplantation genetic diagnosis (PGD) has been applied worldwide for a great variety of single-gene disorders over the last 20 years. The aim of this work was to perform a double-factor preimplantation genetic diagnosis (DF-PGD) protocol in a family at risk for Lynch syndrome. The family underwent a DF-PGD approach in which two blastomeres from each cleavage-stage embryo were biopsied and used for monogenic and comprehensive cytogenetic analysis, respectively. Fourteen embryos were biopsied for the monogenic disease and after multiple displacement amplification (MDA), 12 embryos were diagnosed; 5 being non-affected and 7 affected by the disease. Thirteen were biopsied to perform the aneuploidy screening by short-comparative genomic hybridization (CGH). The improved DF-PGD approach permitted the selection of not only healthy but also euploid embryos for transfer. This has been the first time a double analysis of embryos has been performed in a family affected by Lynch syndrome, resulting in the birth of two healthy children. The protocol described in this work offers a reliable alternative for single-gene disorder assessment together with a comprehensive aneuploidy screening of the embryos that may increase the chances of pregnancy and birth of transferred embryos.

Whole-chromosome aneuploidy analysis in human oocytes: focus on comparative genomic hybridization.

The study of aneuploidy in human oocytes, discarded from IVF cycles, has provided a better understanding of the incidence of aneuploidy of female origin and the responsible mechanisms. Comparative genomic hybridization (CGH) is an established technique that allows for the detection of aneuploidy in all chromosomes avoiding artifactual chromosome losses. In this review, results obtained using CGH in single cells (1PB and/or MII oocytes) are included. The results of oocyte aneuploidy rates obtained by CGH from discarded oocytes of IVF patients and of oocyte donors are summarized. Moreover, the mechanisms involved in the aneuploid events, e.g. whether alterations occurred due to first meiotic errors or germ-line mitotic errors are also discussed. Finally, the incidence of aneuploid oocyte production due to first meiotic errors and germ-line mitotic errors observed in oocytes coming from IVF patients and IVF oocyte donors was assessed.

Reliability of short comparative genomic hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application.

BACKGROUND: Comparative genomic hybridization (CGH) is a valuable alternative to fluorescence in situ hybridization (FISH) for preimplantation genetic screening (PGS) because it allows full karyotype analysis. However, this approach requires the cryopreservation of biopsied embryos until results are available. The aim of this study is to reduce the hybridization period of CGH, in order to make this short-CGH technique suitable for PGS of Day-3 embryos, avoiding the cryopreservation step. METHODS: Thirty-two fibroblasts from six aneuploid cell lines (Coriell) and 48 blastomeres from 10 Day-4 embryos, discarded after PGS by FISH with 9 probes (9-chr-FISH), were analysed by short-CGH. A reanalysis by the standard 72 h-CGH and FISH using telomeric probes was performed when no concordant results between short-CGH and FISH diagnosis were observed. The short-CGH was subsequently applied in a clinical case of advanced maternal age. RESULTS: In 100% of the fibroblasts analysed, the characteristic aneuploidies of each cell line were detected by short-CGH. The results of the 48 blastomeres screened by short-CGH were supported by both 72 h-CGH results and FISH reanalysis. The chromosomes most frequently involved in aneuploidy were 22 and 16, but aneuploidies for the other chromosomes, excepting 1, 10 and 13, were also detected. Forty-one of the 94 aneuploid events observed (43.6%) corresponded to chromosomes which are not analysed by 9-chr-FISH. CONCLUSIONS: We have performed a preliminary validation of the short-CGH technique, including one clinical case, suggesting this approach may be applied to Day-3 aneuploidy analysis, thereby avoiding embryo cryopreservation and perhaps helping to improve implantation rate after PGS.

Transcriptomic profiling of human oocytes: association of meiotic aneuploidy and altered oocyte gene expression.

The ability to identify oocytes with the greatest potential for producing a viable embryo would be of great benefit to assisted reproductive treatments. One of the most important defects affecting oocytes is aneuploidy. Aneuploidy is also closely related with advancing maternal age, a phenomenon not well understood. This study combined a comprehensive cytogenetic investigation of 21 oocytes with a detailed assessment of their transcriptome. The first polar body was removed from all oocytes and aneuploidy assessed using comparative genomic hybridization. Preliminary mRNA transcript data were produced with the use of microarrays for seven of the corresponding oocytes (three normal and four aneuploid). The results obtained for normal and aneuploid oocytes were compared and 327 genes were found to display statistically (P < 0.05) significant differences in transcript levels. Ninety-six of these genes were further assessed in seven aneuploid and seven normal oocytes using real-time PCR. The results indicated that aneuploidy is associated with altered transcript levels affecting a subset of genes. A link between mRNA transcript numbers and age was also observed. The possibility that different transcript levels in the oocyte have an impact on cellular pathways remains to be proven. However, it may be significant that some of the highlighted genes produce proteins involved in spindle assembly and chromosome alignment. Additionally, several genes with altered amounts of transcript produce cell surface or excretory molecules, and could potentially serve as targets for non-invasive oocyte aneuploidy assessment.

D-penicillamine and plasmapheresis in acute liver failure secondary to Wilson's disease.

We report a case of a 19-year-old woman with acute liver failure, Coombs negative hemolytic anemia, and renal failure as initial manifestations of Wilson disease with recovery following medical treatment. The clinical picture and low serum transaminase and alkaline phosphatase levels gave us a clue to suspect Wilson disease and to initiate plasmapheresis and D-penicillamine soon after admission. The serum and urinary copper levels were elevated with low serum ceruloplasmin. We proceeded to ambulatory follow-up with medical treatment with D-penicillamine. A few months later, during the course of a laparoscopic cholecystectomy because of symptomatic gallstone disease, a liver biopsy sample was obtained that showed histological liver fibrosis and strongly elevated levels of liver tissue copper.