How 1 h of abstinence improves sperm quality and increases embryo euploidy rate after PGT-A: a study on 106 sibling biopsied blastocysts.

PURPOSE: The aim of our study was to evaluate the influence of different ejaculatory abstinence time frames (several days versus 1 h) on semen parameters, blastocysts ploidy rate, and clinical results in assisted reproduction cycles on sibling oocytes. METHODS: This is a prospective study including 22 preimplantation genetic testing for aneuploidy (PGT-A) cycles performed between November 2015 and December 2018. Male partners with oligoastenoteratozoospermia produced two semen samples on the day of oocyte retrieval: the first one after several days of abstinence and the second, 1 h after the first one. Oocytes from each patient were divided into two groups: those in group 1 were injected with spermatozoa from the first ejaculate (N = 121) and oocytes in group 2 with spermatozoa from the second one (N = 144). Outcomes of aniline blue test, fertilization, blastocyst formation, ploidy rates, and clinical results were compared between the two groups. RESULTS: Semen volume resulted lower in the second sperm retrieval. Sperm concentration, motility, and morphology were similar in the two groups. A total of 106 blasotcysts were biospied. Higher blastocyst euploidy rates resulted in group 2 (43.6%) than in group 1 (27.5%). A higher percentage of mature chromatine was observed in group 2. CONCLUSION: Using spermatozoa from samples with a shorter abstinence could be a simple method to select higher quality spermatozoa, reducing aneuploidy rate in blastocysts. Prospective randomized controlled trials should be performed to confirm the potential advantage of using semen samples with short abstinence period to improve the outcome of assisted reproduction cycles.

The aging male: Relationship between male age, sperm quality and sperm DNA damage in an unselected population of 3124 men attending the fertility centre for the first time.

OBJECTIVE: the aim of our study was to put forward insights to treat any possible correlation among sperm quality, sperm DNA damage and male age as they may have fertility implications for men who choose to delay fatherhood. MATERIALS AND METHODS: Our study is a non-interventional retrospective analysis of 3124 semen samples from patients that were investigated for the conventional semen parameters. Tunel test assay was set up for the evaluation of the sperm DNA fragmentation index (DFI). We applied the Kappa index to compare both the 1999 and the 2010 World Health Organization (WHO) reference criteria to evaluate the competence of such semen parameters categorization during the standard routine of our laboratory. RESULTS: With regards to our findings, it is possible to underline a significant relationship between aging and semen volume (p = 0.001), motility (p = 0.009), semen viscosity (p < 0.003) and sperm DNA damage (p < 0.009). We found a trend when focusing on the semen concentration (p = 0.05). The analysis of sperm morphology did not show any influence with advancing age (p = 0.606). When comparing both the 1999 and the 2010 WHO scales we found no accordance in the appraisal of sperm morphology but a very good one in the evaluation of the other parameters. CONCLUSIONS: Conventional semen analysis represents the opportunity to draw up a proxy insight on the male fertility status even if semen quality can only indirectly assess the probability of pregnancy. Several studies have verified a decay in the male reproductive system, sperm quality and fertility with advancing age although the reported results are not yet conclusive. Our results substantially agree with those findings outlined in the literature. Moreover we find that the discrepancy between the two WHO reference scales would eventually lead to an improper diagnosis of infertility.

Extent of chromosomal mosaicism influences the clinical outcome of in vitro fertilization treatments.

OBJECTIVE: To assess whether the extent of chromosomal mosaicism can influence the success rate of IVF treatments. DESIGN: Prospective study. SETTING: Private genetic and assisted reproduction centers. PATIENT(S): The transfer of mosaic embryos was offered to 77 women for which IVF resulted in no euploid embryos available for transfer. INTERVENTION(S): All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on day 5/6 of development. Comprehensive chromosome screening was performed using either next-generation sequencing or array-comparative genomic hybridization methodologies. MAIN OUTCOME MEASURE(S): The clinical outcome obtained after transfer of mosaic embryos with low (<50%) and high (≥50%) aneuploidy percentage was compared with that resulting from a control group of 251 euploid blastocysts. RESULT(S): A significantly higher implantation rate (48.9% vs. 24.2%), clinical pregnancy rate/ET (40.9% vs. 15.2%), and live-birth rate (42.2% vs. 15.2%) were observed comparing embryos with mosaicism <50% and ≥50%. Mosaic embryos with high aneuploidy percentage (≥50%) showed a significantly lower clinical pregnancy rate/ET (15.2% vs. 46.4%), implantation rate (24.4% vs. 54.6%), and live-birth rate (15.2% vs. 46.6%) than euploid blastocysts. In contrast, embryos with lower aneuploidy percentage (<50%) have a clinical outcome similar to euploid embryos. CONCLUSION(S): The results of this study further confirm that mosaic embryos can develop into healthy euploid newborns. We demonstrated that the extent of mosaicism influences the IVF success rate. Mosaic embryos with low aneuploidy percentage have higher chances of resulting in the birth of healthy babies compared with embryos with higher mosaicism levels.

Genetic diseases and aneuploidies can be detected with a single blastocyst biopsy: a successful clinical approach.

STUDY QUESTION: Can simultaneous detection of aneuploidies and genetic diseases or chromosomal aberrations in blastocysts reduce the chance of transferring embryos with low implantation potential, guaranteeing good clinical outcomes? SUMMARY ANSWER: The screening for chromosomal aneuploidies revealed that 50.6% of blastocysts diagnosed free of genetic disease or balanced, were aneuploid, therefore avoiding the transfer of blastocysts potentially resulting in implantation failures, miscarriages, or in some cases, in health affected live births. WHAT IS KNOWN ALREADY: PGD is applied in patients at risk of transmitting genetically inheritable diseases to their offspring. It has been demonstrated that aneuploidies can involve chromosomes other than those investigated with PGD, affecting embryo implantation competence. Performing the biopsy at blastocyst level produces higher clinical outcomes allowing a more accurate diagnosis, compared to blastomere biopsy. STUDY DESIGN, SIZE, DURATION: This consecutive case series study was performed from October 2011 to May 2016. Clinical and biological outcomes from 1122 blastocysts obtained in 304 PGD cycles for monogenic diseases (N = 163) or chromosomal rearrangements (N = 141) were analyzed. When the blastocyst resulted transferable after the PGD analysis or chromosomal rearrangement analysis, its ploidy status by mean of preimplantation genetic screening (PGS) was also detected using the same biopsy sample. Mean female age was 35.4 ± 4.2 years old. All biopsies were performed at blastocyst stage and analyzed by Whole Genome Amplification (WGA) followed by PCR for monogenic diseases, and by array-comparative genotype hybridization (array-CGH) for all cycles. PARTICIPANTS/MATERIALS, SETTING, METHOD: All mature oocytes retrieved were injected and cultured individually until the blastocyst stage at 37°C, 6% CO2, 5% O2. When the blastocyst was formed, it was biopsied and vitrified, awaiting the genetic results. The frozen-thawed embryo transfer was performed in a subsequent cycle. In some cases, when the blastocyst was obtained within the morning of Day 5 of culture, it had been maintained in culture and transferred on Day 6, after receiving the genetic report. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 2809 (2718 fresh and 91 frozen-thawed) mature oocytes were injected with a fertilization rate of 75.5% (N = 2120), leading to the development of 2102 embryos. A further 24 frozen embryos, previously vitrified without any genetic testing, were successfully warmed for genetic screening. A total of 2126 embryos were cultured with a blastocyst formation rate of 52.8% (N = 1122); all of them were biopsied from Day 4 to Day 7 of culture. After the genetic analysis, 309 (27.5%) blastocysts resulted transferable, both for monogenic disease or translocation and for their ploidy status, 42 were diploid/aneuploid mosaic, 55 were no result and 716 were not transferable, due to genetic disease or chromosomal rearrangement and/or for their ploidy status. Of note, 316 (50.6% of transferable blastocysts after PGD and 28.2% of total number of biopsied blastocysts) of the blastocysts resulted healthy for the genetic disease or chromosomal rearrangement were aneuploid. Out of 304 PGD/PGS cycles performed, 28.6% (N = 87) resulted in no-transferable blastocysts after only PGD analysis; this percentage increased to 39.8% (N = 121) when also PGS was carried out (Mc Nemar test P < 0.001). A total of 202 embryo-transfers were performed, 53 fresh and 149 cryopreserved, in which 218 healthy or carrier euploid blastocysts were transferred. Clinical pregnancy, implantation and miscarriage rates were 49.0, 47.7 and 9.9%, respectively. To date, 66 deliveries occurred with 70 healthy babies born and 13 pregnancies are still ongoing. Finally, 91 euploid healthy blastocysts are still cryopreserved waiting to be transferred. LIMITATIONS, REASONS FOR CAUTION: A higher than expected cycle cancellation rate could be found due to the double genetic analysis performed. For this reason, particular care should be taken in drafting and explaining informed consent, in order to avoid patient drop out. WIDER IMPLICATIONS OF THE FINDINGS: When the biopsy has to be performed in order to prevent the transmission of an inheritable disease, it should be mandatory to analyze also the genetic status of the blastocyst, avoiding useless embryo-transfers in this particular category of patients. In our study, 316 aneuploid healthy blastocysts could have been transferred without performing PGS, leading to implantation failures, miscarriages, or in some cases, to live births affected by different syndromes. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was obtained for this study. None of the authors have any competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Correlation between aneuploidy, standard morphology evaluation and morphokinetic development in 1730 biopsied blastocysts: a consecutive case series study.

STUDY QUESTION: Are there correlations among human blastocyst ploidy status, standard morphology evaluation and time-lapse kinetics? SUMMARY ANSWER: Correlations were observed, in that euploid human blastocysts showed a higher percentage with top quality inner cell mass (ICM) and trophectoderm (TE), higher expansion grades and shorter time to start of blastulation, expansion and hatching, compared to aneuploid ones. WHAT IS KNOWN ALREADY: Embryo quality has always been considered an important predictor of successful implantation and pregnancy. Nevertheless, knowledge of the relative impact of each morphological parameter at the blastocyst stage needs to be increased. Recently, with the introduction of time-lapse technology, morphokinetic parameters can also be evaluated. However, a large number of studies has reported conflicting outcomes. STUDY DESIGN, SIZE, DURATION: This was a consecutive case series study. The morphology of 1730 blastocysts obtained in 530 PGS cycles performed from September 2012 to April 2014 that underwent TE biopsy and array comparative genomic hybridization was analyzed retrospectively. A total of 928 blastocysts were cultured in a time-lapse incubator allowing morphokinetic parameters to be analyzed. PARTCIPANTS/MATERIALS, SETTING, METHOD: Mean female age was 36.8 ± 4.24 years. Four hunderd fifty-four couples were enrolled in the study: 384, 64 and 6 of them performed single, double or triple PGS cycles, respectively. In standard morphology evaluation, the expansion grade, and quality of the ICM and TE were analyzed. The morphokinetic parameters observed were second polar body extrusion, appearance of two pronuclei, pronuclear fading, onset of two- to eight-cell divisions, time between the two- and three-cell (cc2) and three- and four-cell (s2) stages, morulae formation time, starting blastulation, full blastocyst stage, expansion and hatching timing. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 1730 biopsied blastocysts, 603 were euploid and 1127 aneuploid. We observed that 47.2% of euploid and 32.8% of aneuploid blastocysts showed top quality ICM (P < 0.001), and 17.1% of euploid and 28.5% of aneuploid blastocysts showed poor quality ICM (P < 0.001). Top quality TE was present in 46.5% of euploid and 31.1% of aneuploid blastocysts (P < 0.001), while 26.6% of euploid and 38.1% of aneuploid blastocysts showed poor quality TE (P < 0.001). Regarding expansion grade, 81.1% of euploid and 72.4% of aneuploid blastocysts were fully expanded (Grade 5-6; P < 0.001). The timing of cleavage from the three- to four-cell stage, of reaching four-cell stage, of starting blastulation, reaching full blastocyst stage, blastocyst expansion and hatching were 2.6 (95% confidence interval (CI): 1.7-3.5), 40.0 (95% CI: 39.3-40.6), 103.4 (95% CI: 102.2-104.6), 110.2 (95% CI: 108.8-111.5), 118.7 (95% CI: 117.0-120.5) and 133.2 (95% CI: 131.2-135.2) hours in euploid blastocysts, and 4.2 (95% CI: 3.6-4.8), 41.1 (95% CI: 40.6-41.6), 105.0 (95% CI: 104.0-106.0), 112.8 (95% CI: 111.7-113.9), 122.1 (95% CI: 120.7-123.4) and 137.4 (95% CI: 135.7-139.1) hours in aneuploid blastocysts (P < 0.05 for early and P < 0.0001 for later stages of development), respectively. No statistically significant differences were found between euploid and aneuploid blastocysts for the remaining morphokinetic parameters.A total of 407 embryo transfers were performed (155 fresh, 252 frozen-thawed blastocysts). Higher clinical pregnancy, implantation and live birth rates were obtained in frozen-thawed compared to fresh embryo transfers (P = 0.0104, 0.0091 and 0.0148, respectively). The miscarriage rate was 16.1% and 19.6% in cryopreserved and fresh embryo transfer, respectively. The mean female age was lower in the euploid compared to aneuploid groups (35.0 ± 3.78 versus 36.7 ± 4.13 years, respectively), We found an increasing probability for aneuploidy with female age of 10% per year (odds ratio (OR) = 1.1, 95% CI: 1.1-1.2, P < 0.001). LIMITATIONS, REASONS FOR CAUTION: The main limitation of morphology assessment is that it is a static system and can be operator-dependent. In this study, eight embryologists performed morphology assessments. The main limitation of the time-lapse technology is that it is impossible to rotate the embryos making it very difficult to observe them in case of blastomere overlapping or increased cytoplasmic fragmentation. WIDER IMPLICATIONS OF THE FINDINGS: Although there seems to be a relationship between the ploidy status and blastocyst morphology/development dynamics, the evaluation of morphological and morphokinetic parameters cannot currently be improved upon, and therefore replace, PGS. Our results on ongoing pregnancy and miscarriage rates suggest that embryo evaluation by PGS or time-lapse imaging may not improve IVF outcome. However, time-lapse monitoring could be used in conjunction with PGS to choose, within a cohort, the blastocysts to analyze or, when more than one euploid blastocyst is available, to select which one should be transferred. STUDY FUNDING/COMPETING INTERESTS: No specific funding was obtained for this study. None of the authors have any competing interests to declare.

Comparative genomic hybridization selection of blastocysts for repeated implantation failure treatment: a pilot study.

The aim of this study is to determine if the use of preimplantation genetic screening (PGS) by array comparative genomic hybridization (array CGH) and transfer of a single euploid blastocyst in patients with repeated implantation failure (RIF) can improve clinical results. Three patient groups are compared: 43 couples with RIF for whom embryos were selected by array CGH (group RIF-PGS), 33 couples with the same history for whom array CGH was not performed (group RIF NO PGS), and 45 good prognosis infertile couples with array CGH selected embryos (group NO RIF PGS). A single euploid blastocyst was transferred in groups RIF-PGS and NO RIF PGS. Array CGH was not performed in group RIF NO PGS in which 1-2 blastocysts were transferred. One monoembryonic sac with heartbeat was found in 28 patients of group RIF PGS and 31 patients of group NO RIF PGS showing similar clinical pregnancy and implantation rates (68.3% and 70.5%, resp.). In contrast, an embryonic sac with heartbeat was only detected in 7 (21.2%) patients of group RIF NO PGS. In conclusion, PGS by array CGH with single euploid blastocyst transfer appears to be a successful strategy for patients with multiple failed IVF attempts.

Impact of oocyte cryopreservation on embryo development.

OBJECTIVE: To verify whether the morphologic evaluation of zygotes and embryos derived from thawed oocytes could provide some relevant information regarding their developmental performance. DESIGN: Fertilization, zygote, and embryo morphology from sibling fresh and frozen oocytes was compared. SETTING: Reproductive Medicine Unit, Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. PATIENT(S): Two hundred thirty-four patients underwent intracytoplasmic sperm injection cycles from which 1,101 spare metaphase II oocytes were cryopreserved. Subsequently, 256 thawing cycles were performed, and 997 oocytes were thawed. INTERVENTION(S): Intracytoplasmic sperm injection was performed on both fresh and frozen oocytes. MAIN OUTCOME MEASURE(S): Fertilization rates, pronuclear zygote morphology, and embryo cleavage rates. RESULT(S): Thawed oocytes had lower chances of being fertilized and developing into top-quality zygotes and regularly cleaving embryos when compared with sibling fresh oocytes irrespective of female age. As a result, the percentage of transferred cycles was significantly lower in frozen cycles compared with fresh cycles (79% and 93%, respectively); the proportion of transferred top-quality embryos followed the same trend. CONCLUSION(S): Reduced fertilization and cleavage rates in frozen cycles when compared with sibling fresh oocytes suggest that, even if surviving thawing, the process of slow freezing has a negative impact on the potential of further growth that is evident as early as the first cleavage divisions.

Embryo morphology and development are dependent on the chromosomal complement.

OBJECTIVE: To analyze embryo morphology in relation to the corresponding chromosomal status, in order to evaluate the effects of aneuploidy over fragmentation, delayed cleavage, and arrested cleavage. DESIGN: Retrospective study. SETTING: Reproductive Medicine Unit, Società Italiana di Studi di Medicina della Riproduzione, Bologna, Italy. PATIENT(S): A total of 662 patients with a poor prognosis for pregnancy underwent 916 cycles of preimplantation genetic diagnosis for aneuploidy. INTERVENTION(S): The chromosomes XY, 13, 15, 16, 18, 21, and 22 were analyzed in blastomeres biopsied from day 3 embryos. MAIN OUTCOME MEASURE(S): Embryo morphology, chromosomal status of the analyzed blastomeres, and spreading and reanalysis of nontransferred embryos. RESULT(S): The incidence of chromosomal abnormalities was significantly higher in arrested or slow-cleaving embryos, and in embryos cleaving too fast, compared to embryos with eight cells at 62 hours after insemination. The presence of an uneven number of blastomeres or fragments scattered in the perivitelline space was associated with an increased incidence of chromosomal abnormalities. CONCLUSION(S): A correlation between embryo development and chromosomal complement makes the incidence of chromosomal abnormalities significantly higher in embryos dividing according to a time frame and a symmetry plan which are different from what are expected. The type of fragmentation is also related to chromosomal status, which explains why the extrusion of fragments might severely affect embryo viability.