Oocyte vitrification does not affect early developmental timings after intracytoplasmic sperm injection for women younger than 30 years old.

Oocyte vitrification causes a temporary disassembly of the metaphase plate and spindle, which needs time to recover after warming. As a consequence, early post-fertilization events-such as timing of second polar body extrusion-might be altered, with unknown effects on preimplantation development, timing to pronuclear breakdown, and timing of cleavages. The aim of this study was to evaluate if differences exist among these events when comparing embryos obtained from fresh-donated versus vitrified/warmed oocytes from young women. We performed a prospective study with 201 embryos from 100 fresh and 101 vitrified/warmed oocytes that were subsequently fertilized by intracytoplasmic sperm injection. Kaplan-Meier curves of each time period were generated, in which we observed that median developmental times did not differ between embryos from fresh versus vitrified/warmed oocytes among all the metrics assessed. Thus, for young women without fertility problems, no differences exist between the timing of early developmental milestones in embryos derived from fresh or vitrified oocytes, and vitrification does not affect the preimplantation development of the resulting embryos. Mol. Reprod. Dev. 83: 624-629, 2016. © 2016 Wiley Periodicals, Inc.

Semen residual viral load and reproductive outcomes in HIV-infected men undergoing ICSI after extended semen preparation.

The aim of this study was to evaluate the residual presence of the human immunodeficiency virus (HIV) following a triple gradient extended semen wash from ejaculates of serodiscordant couples, and analyse their reproductive outcomes after intracytoplasmic sperm injection (ICSI). For this purpose, a retrospective analysis of our database was performed in serodiscordant couples, with HIV-infected men and non-infected women, using fresh or frozen sperm with ICSI in oocytes from either the patients or donors from January 2006 to September 2013. Overall, the rate of positive HIV test after semen washing was 1.86%. The positive beta human chorionic gonadotrophin, clinical and ongoing pregnancy rates in patients with their own oocytes were 47.1%, 37.5% and 30.8%, respectively, and 58.6%, 50.8% and 39.1%, respectively, in oocyte donation cycles. To summarize, the described method of sperm washing based on triple gradient sperm selection coupled with extensive centrifugations is a highly reliable technique for HIV removal, as it provides lower than reported post-wash positive tests while maintaining high pregnancy rates in assisted reproduction cycles. Despite extensive personnel training and effectiveness of the washing protocol, post-wash HIV test on semen is recommended to identify residual positive samples.

Are we ready to inject? Individualized LC-CUSUM training in ICSI.

PURPOSE: The objective of this prospective, single center study was to develop a personalized training scheme for intracytoplasmic sperm injection (ICSI) through the use of learning curve-cumulative summation (LC-CUSUM), which allows to tailor training to the trainee performance, and to validate it against the performance of experienced embryologists. METHODS: Five trainees microinjected latex microspheres (LM) into vitro matured oocytes. A microinjection was considered successful when the oocyte did not lyse in the 24 h following the injection. RESULTS: Each trainee became proficient at ICSI after a variable number of injections, ranging from 35 to 80. Trainees that achieved proficiency went on to perform ICSI with human gametes in a clinical setting with proficiency comparable to that of experienced embryologists. CONCLUSIONS: We show that LC-CUSUM based personalized ICSI training is feasible and allows trainees to be as proficient as trained embryologists when treating actual patients.

Oxidative stress level in fresh ejaculate is not related to semen parameters or to pregnancy rates in cycles with donor oocytes.

PURPOSE: The purpose of the present study is to study the relationship between oxidative stress (OS) in semen, semen characteristics, and reproductive outcomes in oocyte donation intracytoplasmic sperm injection (ICSI) cycles. METHODS: OS was measured in 132 semen samples. RESULTS: OS levels were as follows: very high (1.5 %), high (43.2 %), low (30.3 %), and very low (25.0 %). Overall seminal parameters were as follows: volume (ml) = 4.2 (SD 2.1), concentration (millions/ml) = 61.6 (SD 59.8), motility (a+b%) = 47.4 (SD 18.0), and normal spermatozoa (%) = 8.2 (SD 5.1). Of the 101 cycles that reached embryo transfer, 55.4 % evolved in biochemical, 46.5 % in clinical, and 43.6 % in ongoing pregnancy. OS level does not relate to seminal parameters, fertilization rate, or pregnancy outcomes. CONCLUSIONS: OS testing by nitro blue tetrazolium (NBT) in fresh ejaculate might not be useful for all patients. Reproductive results with young oocytes and ICSI do not seem to be affected by OS-level semen.

Double-factor preimplantation genetic diagnosis: monogenic and cytogenetic diagnoses analyzing a single blastomere.

OBJECTIVE: Enhancing implantation rates in preimplantation genetic diagnosis (PGD) cycles is still a challenging aspect to address. As aneuploidy can be one of the factors influencing the low implantation rates obtained, the aim of this work was to combine monogenic analysis with comprehensive aneuploidy screening (double factor) in order to transfer the selected (healthy and euploid) embryos in the same in-vitro fertilization (IVF) cycle. METHOD: In the present double-factor PGD (DF-PGD) approach, a single blastomere was biopsied from each embryo, and the whole genome amplification DNA product obtained was successfully used for both monogenic analysis and metaphase comparative genomic hybridization cytogenetic screening. The developed DF-PGD was applied to 62 embryos from seven families at risk for monogenic-inherited diseases in a total of seven IVF-DF-PGD cycles. RESULTS: While 68.2% of the diagnosed embryos were healthy for the monogenic diseases, only 43.3% of them were chromosomally normal considering aneuploidies and/or segmental chromosome imbalances. Six out of seven families had transferrable embryos according to DF-PGD results. Two healthy babies were born from the 11 selected embryo transfers. CONCLUSION: In families at risk for monogenic diseases, the DF-PGD is a useful tool to select healthy and potentially viable embryos for transfer, according to their chromosome complement.

Detection of DNA damage in cumulus cells using a chromatin dispersion assay.

DNA damage in cumulus cells (CCs) might be related with the developmental competence of the enclosed oocytes, however, conclusive studies are missing, partially due to the lack of a reliable, cheap, fast, and reproducible DNA damage test. We report the development of a chromatin dispersion test that allows for a fast evaluation of double strand DNA (ds-DNA) damage in CCs. The whole experiment was performed using CCs from 103 oocyte retrieval cycles evaluating the prototype D3-MAX ability (a chromatin dispersion based assay) to detect DNA breaks against in situ nick translation (ISNT) and a two tailed comet assay (TT-comet). Samples were collected from women younger than 35 years of age with a good response to stimulation. Pooled cumulus cells of MII oocytes were used. The chromatin dispersion assay results correlate with the double strand-DNA breaks values assessed by the TT-comet assay (Spearman Rho = 0.624; p = 0.003;), while the correlation was poor when compared to the single strand DNA (ss-DNA) breaks observed also with the TT-comet assay (Spearman Rho = -0.141; p = 0.554). ISNT showed a correspondence in the same cells between enzymatic incorporation of modified nucleotides and halos of chromatin dispersion. We conclude that D3-Max test detects mainly ds-DNA breaks in cumulus cells and is a reliable, fast, and easy reproducible assay suitable for routine clinical practices once the influence on oocyte quality has been established.

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.

Detection of unbalanced chromosome segregations in preimplantation genetic diagnosis of translocations by short comparative genomic hibridization.

OBJECTIVE: To apply a comprehensive chromosomal screening through short comparative genomic hybridization (CGH) in the preimplantation genetic diagnosis (PGD) of translocations. DESIGN: Clinical research study. SETTING: A PGD laboratory and two IVF clinics. PATIENT(S): Three Robertsonian translocation carriers, two reciprocal translocation carriers, and a double-translocation carrier. INTERVENTION(S): After using the short-CGH approach in the reanalysis of two unbalanced embryos, discarded from a PGD for a reciprocal translocation carrier, the same method was applied in the PGD of day-3 embryos of translocation carriers. MAIN OUTCOME MEASURE(S): Ability of short CGH to detect partial chromosomal abnormalities in unbalanced embryos, translocation segregation proportions, and proportion of embryos carrying chromosomal abnormalities not related to the translocations. RESULT(S): The short-CGH technique detected errors resulting from the meiotic segregation of the chromosomes involved in the translocations and other abnormalities affecting the remaining chromosomes. Alternate segregation was detected most frequently among Robertsonian translocation cases, whereas unbalanced chromosome segregations were found predominantly in reciprocal ones. Aneuploidy and structural chromosome errors were found more frequently in Robertsonian than in reciprocal translocation carriers. Application of short-CGH PGD achieved pregnancy in two cases. CONCLUSION(S): Short CGH is a reliable approach for PGD of translocations, as it is capable of detecting partial chromosome errors caused by unbalanced segregations simultaneously to the screening of all chromosomes, and it may improve the results after PGD for translocation carriers.

Transvaginal versus transabdominal ultrasound guidance for embryo transfer in donor oocyte recipients: a randomized clinical trial.

OBJECTIVE: To compare pregnancy and implantation rates with transvaginal (TV) versus transabdominal (TA) ultrasound-guided embryo transfer (ET). DESIGN: Randomized, clinical trial registered at clinicaltrials.gov (NCT 01137461). SETTING: Private, infertility clinic. PATIENT(S): Three-hundred thirty randomized recipients of donor oocytes. INTERVENTION(S): Embryo transfer using TV (with empty bladder, using the Kitazato ET Long catheter) versus TA ultrasound guidance (with full bladder, using the echogenic Sure View Wallace catheter). MAIN OUTCOME MEASURE(S): Overall pregnancy, clinical pregnancy, implantation, and ongoing pregnancy rates. Duration and difficulty of ET. Patient-reported uterine cramping and discomfort, as evaluated by questionnaire. RESULT(S): No statistically significant differences were observed in clinical pregnancy 50.9% versus 49.4% (95% confidence interval of the difference: -9.2 to +12.2%), implantation 34.5% versus 31.4% (95% CI of the difference: -4 to +10.3%) between the TV and TA ultrasound-guided groups. Transfer difficulty (6% versus 4.2%) and uterine cramping (27.2% versus 18.3%) were not statistically significantly different between treatment groups. Total duration (154±119 versus 85±76 seconds) was statistically significantly higher in the TV ultrasound group. Light to moderate-severe discomfort related to bladder distension was reported by 63% of the patients in the TA ultrasound group. CONCLUSION(S): Transvaginal ultrasound-guided ET yielded similar success rates compared with the TA ultrasound-guided procedure without requiring the assistance of a sonographer. It was associated with increased patient comfort due to the absence of bladder distension.

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.

Errors at mitotic segregation early in oogenesis and at first meiotic division in oocytes from donor females: comparative genomic hybridization analyses in metaphase II oocytes and their first polar body.

The aim of this work is to analyze, using the comparative genomic hybridization technique, the frequencies and the mechanisms involved in the production of aneuploidy events in donor oocytes. The results showed that 32.1% of them were aneuploid, with 51.7% of those originating from first meiotic division errors and 48.3% from the presence of aneuploid oogonium.

Outcome of twin babies free of Von Hippel-Lindau disease after a double-factor preimplantation genetic diagnosis: monogenetic mutation analysis and comprehensive aneuploidy screening.

OBJECTIVE: To increase the embryo implantation rate, a double-factor preimplantation genetic diagnosis (DF-PGD) was performed, selecting for transfer potentially euploid evolved embryos free of the mutation responsible for Von Hippel-Lindau syndrome (VHL). DESIGN: Case report. SETTINGS: Medical university center and a private IVF center. PATIENT(S): A patient carrier of the R161Q mutation on the VHL gene. INTERVENTION(S): After first polar body (1PB) biopsy, it was analyzed using comparative genomic hybridization (1PB-CGH). On day +3, mutation detection using minisequencing and short tandem repeat analysis was performed in multiple displacement amplification products of a single blastomere per embryo. MAIN OUTCOME MEASURE(S): Transfering embryos free of the disease and originating from euploid oocytes. RESULT(S): Nine of the twelve oocytes obtained were successfully analyzed using 1PB-CGH. One of them was aneuploid (1PB #1: 29XX,+2,+10,+12,+17,+19), and the rest were euploid. All of the oocytes were fertilized and became evolved embryos. Six of the embryos were VHL unaffected and had good quality. Five (83%) of them were potentially euploid. According to cytogenetic results, two of the evolved and healthy embryos were transfered, achieving the birth of healthy twin babies. CONCLUSION(S): The DF-PGD can be a useful tool to increase implantation of transfered embryos in PGD for monogenic diseases.

Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: case report.

Preimplantation genetic diagnosis (PGD) for monogenic diseases is widely applied, allowing the transfer to the uterus of healthy embryos. PGD is also employed for the detection of chromosome abnormalities for couples at high risk of producing aneuploid embryos, such as advanced maternal (>35 years). A significant number of patients requesting PGD for monogenic diseases are also indicated for chromosome testing. We optimized and clinically applied a PGD protocol permitting both cytogenetic and molecular genetic analysis. A couple, carriers of two cystic fibrosis (CF) mutations (c.3849 + 10 KbC > T and c.3408C > A) with a maternal age of 38 years and two previously failed IVF-PGD cycles, was enrolled in the study. After ovarian stimulation, six oocytes were obtained. To detect abnormalities for all 23 chromosomes of the oocyte, the first polar body (1PB) was biopsied from five of the oocytes and analyzed using comparative genomic hybridization (CGH). CGH analysis showed that 1PB 1 and 1PB 4 were aneuploid (22X,-9,-13,+19 and 22X,-6, respectively), while 1PB 2, 1PB 3 and 1PB 6 were euploid. Blastomere biopsy was only applicable on embryos formed from Oocyte 3 and Oocyte 6. After whole-genome amplification with multiple displacement amplification, a multiplex PCR, amplifying informative short tandem repeats (D7S1799; D7S1817) and DNA fragments encompassing the mutation sites, was performed. MiniSequencing was applied to directly detect each mutation. Genetic diagnosis showed that Embryo 6 was affected by CF and Embryo 3 carried only the c.3849 + 10 KbC > T mutation. Embryo 3 was transferred achieving pregnancy and a healthy boy was born. This strategy may lead to increased pregnancy rates by allowing preferential transfer of euploid embryos.