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Which factors influence the success rate of egg donation programmes with imported vitrified oocytes?Observational longitudinal cohort study of 431 oocyte donation cycles conducted between January 2015 and February 2019. A total of 398 couples underwent an IVF cycle with imported donated vitrified eggs. All consecutive oocyte donation cycles conducted at the Centre for Reproductive Medicine of the European Hospital in Rome, Italy, were included.A univariable analysis was conducted. Among the demographic characteristics studied, body mass index (BMI) was significantly able to influence outcome. In 49% of unsuccessful cycles, men were overweight, compared with 39.4% in cycles with a positive beta-HCG test (P = 0.03). The relationship between female or donor BMI and treatment outcome, however, was not statistically significant. The day on which the embryo is transferred affects the outcome of the cycle: transferring on day 2 versus day 5 reduces the probability of clinical pregnancy. The relationship between male BMI classes and semen parameters was studied: obese men have a significantly lower sperm concentration than normal-weight men (P = 0.006 after Bonferroni correction). No statistically significant differences were found in semen volume (P = 0.722), sperm morphology (P = 0.100) and motility (P = 0.179) in obese men compared with normal weight men.In addition to the number of oocytes available after warming, male BMI, semen characteristics and blastocyst transfer are critical parameters able to influence the reproductive outcomes of egg donation programmes with imported vitrified oocytes.

SummaryThe aim of our study was to evaluate the correlation between sperm quality and ploidy status of the derived blastocysts. We performed a retrospective analysis on a restricted pool of patients enrolling only those who had no female factors. Male patients with genetic factors affecting spermatogenesis were also excluded. We chose a maternal age ≤38 years to decrease the female factor, therefore the male factor was the main component of sterility. We divided the patients in four groups based on semen quality and comparing fertilization, pregnancy and euploidy rates above all. In total, 201 intracytoplasmic sperm injection (ICSI) cycles were enrolled in the study. Cycles were divided into four groups, according to semen source: normal semen, oligoasthenoteratozoospermia (OAT), cryptospermia or non-obstructive azoospermia (NOA). An extremely statistically lower fertilization rate was found in NOA patients. Unexpectedly, no differences were detected in blastocyst formation, euploidy, aneuploidy and mosaicism rates among the four groups. Interestingly, we also found a higher abortion rate comparing NOA to normal semen with an odds ratio of 4.67. In our study no statistically significant differences among the analyzed groups were found, showing little or no effect at all using spermatozoa from different semen sources or quality. This may be linked to the oocyte competence of fixing sperm DNA damage and it could be hypothesized that only sperm with a good rate of DNA integrity are able to fertilize the oocyte, explaining why poor quality semen is reflected in a low fertilization rate without effect on ploidy.

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.

Background: Preimplantation genetic testing (PGT) is widely used today in in-vitro fertilization (IVF) centers over the world for selecting euploid embryos for transfer and to improve clinical outcomes in terms of embryo implantation, clinical pregnancy, and live birth rates. Methods: We report the current knowledge concerning these procedures and the results from different clinical indications in which PGT is commonly applied. Results: This paper illustrates different molecular techniques used for this purpose and the clinical significance of the different oocyte and embryo stage (polar bodies, cleavage embryo, and blastocyst) at which it is possible to perform sampling biopsies for PGT. Finally, genetic origin and clinical significance of embryo mosaicism are illustrated. Conclusions: The preimplantation genetic testing is a valid technique to evaluated embryo euploidy and mosaicism before transfer.

The goal of this study is to evaluate MYOInositol effects on spermatozoa motility, in patients’ ejaculates with severe varicocele or hyper viscosity. The study included normal viscosity ejaculate from 30 patients affected by varicocele and hyper viscosity ejaculate from 33 patients without any testicular pathologies. All selected samples showed sperm concentration > 2 million/ml and progressive motility < 32%. In both groups, the pellet obtained after centrifugation in buffered medium, was divided in two aliquots, both incubated for 15 minutes at 37°C: one with MYO-Inositol and the other one, as control, only in phosphate buffered saline (PBS). Afterwards, the sperm progressive motility was assessed using Computer Assisted Sperm Analysis (CASA system). Incubation with MYO-Inositol improved sperm progressive motility in high viscosity samples compared to control group (38.9% ± 3.0 vs 24.35% ± 2.41, respectively; p ≤ 0.0001). Conversely, no statistically significant difference was observed in total sperm progressive motility in varicocele samples compared with control group (22.7% ± 2.07 vs 26.7% ± 3.31, respectively; p = 0.085). The MYO-Inositol positive effect on spermatozoa motility may depend on the type of sperm damage: heavy structural and biochemical defects which typically affects patients with varicocele are not restored by Inositol. On the contrary, MYOInositol is able to improve sperm motility in semen samples with high viscosity, since those samples show no substantial structural sperm defects.

Introduction Chromosomal mosaic embryos are characterized by the presence of chromosomally different cell lines within the same embryo. In a recent published study, we have demonstrated that mosaic embryos hold the potential to implant and result in the birth of healthy babies. Therefore, the transfer of these embryos is now offered as an option for women who undergo in vitro fertilization (IVF) resulting in only mosaic embryos but no euploid embryos for transfer. However, there is still few data concerning the impact of mosaicism on viability, and the classification of such embryos in relation with their reproductive potential is unclear. Here we investigated if chromosomal constitution of mosaic embryos influences the clinical outcome of in vitro fertilization treatments Material and methods The transfer of mosaic embryos was offered to 200 women for whom IVF had resulted in no euploid embryos between May 2016-May 2018. Trophectoderm (TE) biopsy was performed on Day-5 of development or Day6/7 for slow growing embryos. The clinical outcome obtained after transfer of mosaic embryos with different chromosomal constitution was compared with each other and with that obtained from a control group of 500 euploid blastocysts. Comprehensive chromosome testing PGT-A was performed using high resolution next generation sequencing (NGS) methodology. TE biopsies were classified as mosaic if they had 20%-80% abnormal cells. For statistical analysis mosaic embryos were divided in four groups based on chromosomal constitution: mosaic monosomy (single and double monosomies; MM), mosaic trisomy (single and double trisomies, MT), mosaic complex aneuploidy (more than two different aneuploidies; MC) and mosaic segmental aneuploidy (single and double deletion or insertion >5Mb, MS). Results MM showed significant higher birth rate compared to MT (46% vs 24%,p=0.02), MC (46% vs 23%,p=0.03) and MS (46% vs 22%,p=0.02). No significant difference was observed in clinical outcome between the groups MT, MC and MS. A comparison of the clinical outcomes with control euploid group showed no significant difference between euploid control and MM, while a significant low implantation rate (55.4% vs 37% MT, vs 31% MC, vs 23% MS,p Conclusion The study demonstrated that embryos with MT, MS and MC aneuploidies have lower chances of resulting with birth of healthy babies compared to embryos with MM. More interestingly, no difference has been find in clinical results between MM and euploid control group. These findings should be considered for genetic counseling.

Can next-generation sequencing (NGS) techniques be used reliably for comprehensive aneuploidy screening of human embryos from patients undergoing IVF treatments, with the purpose of identifying and selecting chromosomally normal embryos for transfer?Extensive application of NGS in clinical preimplantation genetic screening (PGS) cycles demonstrates that this methodology is reliable, allowing identification and transfer of euploid embryos resulting in ongoing pregnancies.The effectiveness of PGS is dependent upon the biology of the early embryo and the limitations of the technology. Fluorescence in situ hybridization, used to test for a few chromosomes, has largely been superseded by microarray techniques that test all 24 chromosomes. Array comparative genomic hybridization (array-CGH) has been demonstrated to be an accurate PGS method and has become the de facto gold standard, but new techniques, such as NGS, continue to emerge.The study consisted of a prospective trial involving a double blind parallel evaluation, with both NGS and array-CGH techniques, of 192 blastocysts obtained from 55 consecutive clinical PGS cycles undertaken during the period of September to October 2013. Consistency of NGS-based aneuploidy detection was assessed by matching the results obtained with array-CGH-based diagnoses. Primary outcome measure was accuracy of the chromosomal analysis; secondary outcome measures were clinical outcomes.Fifty-five patients (median age 39.3 years, range 32-46) undergoing PGS were enrolled in the study. All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on Day 5 of development or Day 6/7 for slower growing embryos. The method involved whole genome amplification followed by both NGS and array-CGH. The MiSeq control software, real-time analysis and reporter performed on-board primary and secondary bioinformatics analysis. Copy number variation analysis was accomplished with BlueFuse Multi software.A total of 192 blastocysts were blindly evaluated with the NGS-based protocol. Paired comparison between NGS and array-CGH from individual embryos showed concordant results in 191/192 (99.5%) of the blastocysts tested. In total 4608 chromosomes were assessed, 211 (4.6%) of which carried a copy number imbalance. NGS specificity for aneuploidy calling (consistency of chromosome copy number assignment) was 99.98% (4333/4334; 95% confidence interval [95% CI]: 99.87-100) with a sensitivity of 100% (211/211, 95% CI: 99.25-100). Despite one discordant result, NGS specificity and sensitivity for aneuploid embryo calling (24-chromosome diagnosis consistency) were both 100% since the discordant sample presented several other aneuploidies. Clinical application of the NGS-based approach revealed 74/192 (38.5%) euploid blastocysts. Following transfer of 50 embryos in 47 women, 34 women had positive hCG levels: 30 pregnancies continued, confirmed by at least one fetal sac and heart beat (63.8% clinical pregnancy rate/embryo transfer), 3 were biochemical and 1 miscarried. A total of 32 embryos implanted and led to the presence of a fetal sac (64.0% implantation rate). All pregnancies went to term resulting in the birth of 31 healthy babies.Although clinical results reported high pregnancy outcomes following transfer of screened embryos, further data and broad-based clinical application are required to better define the role of NGS in PGS. Before recommending widespread application, a randomized controlled trial confirming its clinical effectiveness is advisable.This is the first study reporting extensive application of NGS-based comprehensive aneuploidy screening on embryos at blastocyst stage in a clinical setting versus array-CGH as test of reference. NGS has demonstrated a reliable methodology, with the potential to improve chromosomal diagnosis on embryos especially in terms of high-throughput, automation and ability to detect aneuploidy. NGS methodology may represent a valuable alternative to the other comprehensive aneuploidy screening techniques currently available.No external funding was sought for this study. Drs F.K. and C.-E.M. are full-time employees of Illumina, Inc., which provided NGS library and sequencing reagents for the study. All other authors have no conflicts to declare.Not applicable.

To evaluate the role of co-transfer of embryos derived from vitrified oocytes accumulated during the previous modified natural cycles and an embryo developed from the last one as an alternative to repetitive single embryo transfer in a fresh modified natural cycle. Thirty-six patients underwent ICSI procedure with three frozen natural oocytes supplemented by a fresh one obtained from the fourth modified natural cycle. Thirty-one controls received at least three consecutive single embryo transfer in a fresh modified natural cycle. In the study group the oocyte retrieval, survival and total fertilization rate were 73.0 %, 78.1 %, and 64.5 %, respectively. Fifty-two embryos were transferred in 29 transfers. In the control group the oocyte retrieval and fertilization rate was 77.4 % and 83.7 %, respectively. Fifty single embryo transfers were performed. Of a total 14 pregnancies obtained in the study group 10 were defined as clinical and 4 as abortions. In the control group a total of 8 single clinical pregnancies and 2 miscarriages were encountered. The overall (20.0 % vs 48.2 %) and the clinical (16.0 % vs 34.4 %) pregnancy rate were significantly higher in the study group having cumulative embryo transfer following the oocyte accumulation. These data demonstrate that the co-transfer of embryos derived from vitrified oocytes accumulated during the previous modified natural cycles and an embryo developed from the last fresh modified natural cycle assure an excellent clinical outcome with the overall and clinical pregnancy rate significantly higher compared to the repetitive single embryo transfer in a fresh modified natural cycle.

Objective To evaluate the efficiency of slush nitrogen vitrification of human oocytes with or without cumulus cells in terms of survival rate and maintenance of meiotic spindle. Design Randomized, comparative study. Setting Medical center. Patient(s) A total of 274 oocytes obtained from 46 couples undergoing infertility treatment. Intervention(s) Metaphase II oocytes were divided into groups A and B, vitrified with and without cumulus cells, respectively. Main Outcomes Measure(s) Survival rates and maintenance of meiotic spindle observed immediately after warming and 3 hours after incubation. Result(s) No statistically significant difference was detected between the two groups in terms of survival rate, but a significantly higher percentage of detectable spindle was observed in group B (completely denuded oocytes), either immediately after warming or 3 hours after incubation. Conclusion(s) Complete denudation of oocytes before slush nitrogen vitrification does not influence survival rates but positively affects oocyte meiotic spindle competence. These data support the hypothesis that cumulus cells during vitrification represent an obstacle to cryoprotectant penetration more than having a protective role for the oocyte.

Italian legislation regarding reproductive medicine limits the number of embryos transferred per attempt to three. Thus, in order to achieve pregnancy, more IVF cycles may be required, generating a need for methods of ovarian stimulation with fewer side effects. The gonadotrophin-releasing hormone (GnRH) antagonists have several advantages in this respect, but there is a debate regarding a possible lower pregnancy rate from resulting cycles. This study evaluated the clinical applicability of GnRH antagonists for ovarian stimulation in young women undergoing intracytoplasmic sperm injection (ICSI) in which only three oocytes can be fertilized. The 200 women treated with GnRH antagonist had a significantly shorter stimulation and lower gonadotrophin consumption, oestradiol concentration, total and mature oocyte recovery as compared with 200 matched controls treated with GnRH agonist. No differences were found between the groups in the number of normal zygotes, total cleaved, transferred and high quality embryos, or in the clinical outcomes. Thus, the previously reported lower pregnancy rate in GnRH antagonist cycles may be related to the oocyte characteristics. Finally, under conditions of oocyte number restriction, the GnRH antagonist-based cycles may be proposed as an efficacious, safe and minimally invasive alternative to GnRH agonist in a standard long protocol.

Over the last two decades, easier and less expensive stimulation treatments have been largely replaced by more complex and more demanding protocols. Since the mid-nineties, long-term gonadotrophin-releasing hormone agonist stimulation protocols have been widely used. Such lengthy expensive regimens are not free from short- and long-term risks and complications. Mild stimulation protocols reduce the mean number of days of stimulation, the total amount of gonadotrophins used and the mean number of oocytes retrieved. The proportion of high quality and euploid embryos seems to be higher compared with conventional stimulation protocols and the pregnancy rate per embryo transfer is comparable. Moreover, the reduced costs, the better tolerability for patients and the less time needed to complete an IVF cycle make mild approaches clinically and cost-effective over a given period of time. However, further prospective randomized studies are needed to compare cumulative pregnancy rates between the two protocols. Natural cycle IVF, with minimal stimulation, has been recently proposed as an alternative to conventional stimulation protocols in normo- and poor responder patients. Although acceptable results have been reported, further large prospective randomized studies are needed to better evaluate the efficacy of these minimal regimens compared with conventional stimulation approaches.

Implantation of 18 mosaic aneuploid blastocysts obtained through in vitro fertilization into 18 women resulted in six pregnancies of normal karyotype. These pregnancies proceeded to term, with the birth of six healthy babies.

A computer-assisted polarization microscopy system (polscope) has made it possible to analyse the meiotic spindle of oocytes subjected to intracytoplasmic sperm injection (ICSI) without affecting their viability. It has been shown that the presence of a detectable birefringent meiotic spindle inside the oocyte cytoplasm of human metaphase II (MII) prepared for ICSI is an indicator of oocyte quality, such as fertilization and developmental ability. Meiotic spindle imaging has also shown that this structure, when detectable, is not always aligned with the first polar body (PB1) in fresh MII oocytes. The relationship between the degree of meiotic spindle deviation from the PB1 location and ICSI outcomes is discussed in this paper. When the meiotic spindle of in-vitro matured oocytes is analysed, it is always found to be aligned with the PB1, suggesting that the misalignment observed in the oocytes matured in vivo results from the PB1 displacement during the manipulations for the cumulus and corona removal. Furthermore, polscope analysis of meiotic spindle changes in living MII oocytes subjected to freezing and thawing procedures has shown that the current techniques of oocyte cryopreservation cause meiotic spindle destruction. The polscope system may assist in the selection of fresh and thawed oocytes for ICSI.

BACKGROUND: The clinical efficacy of the current methods used for cryopreservation of metaphase II human oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation. METHODS: Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2-propanediol with 0.1 M sucrose using a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at sequential steps of the freezing and thawing procedures with the use of a computer-assisted polarization microscopy system (Polscope). RESULTS: The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37∞C in culture medium. CONCLUSIONS: The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction.

Does the health status of infants fathered by nonmosaic Klinefelter syndrome (KS) patients whose partners underwent ICSI with sperm obtained from testicular dissection reveal any genetic risk for the offspring?.KS patients undergoing testicular sperm extraction (TESE) are capable of conceiving healthy children.Paternity has been successfully achieved in nonmosaic KS patients (47,XXY karyotype) by ICSI using either ejaculated or testicular spermatozoa. A crucial concern is the potential transmission of genetic abnormalities to the offspring. Some studies reported that 47,XXY spermatogonia are capable of completing spermatogenesis leading to the production of mature spermatozoa with increased aneuploidies. Other authors showed that where focal spermatogenesis is present in nonmosaic KS males, it originates from euploid germ cells and, therefore, produces normal mature gametes. In support of this finding, at present, the great majority of children born from nonmosaic KS patients are chromosomally normal.From April 2004 to June 2010, 38 azoospermic patients with nonmosaic KS were examined for the presence of testicular spermatozoa. Spermatozoa were retrieved from 15 patients and 26 ICSI cycles were done (16 with cryopreserved sperm). There were 15 pregnancies leading to the birth of 16 babies who were karyotyped at amniocentesis and after birth.Participants were recruited from couples attending the European Hospital, Rome, and Clinica MARGen, Granada, for infertility treatment. Both the European Hospital and Clinica MARGen are private clinics. Testicular tissue was extracted with TESE or micro-TESE. After retrieval, fresh sperm was used for ICSI or it was cryopreserved for future use.Spermatozoa were retrieved from 15 patients (14 TESE and 1 micro-TESE) out of 38 (39.5%). A total of 26 ICSI cycles were performed: 10 with fresh and 16 with cryopreserved-thawed sperm. Mean ages (y) of patients with positive and negative sperm retrieval were, respectively, 34.8 ± 1.72 and 35.6 ± 4.08 (NS, nonsignificant). Comparing ICSI cycles performed with fresh sperm (n = 10) to those performed with frozen-thawed sperm (n = 16): Fertilization rates per injected oocyte were 53.0% (44 of 83) and 47.8% (32 of 67), respectively (NS). The cleavage rate per injected oocyte was 90.6% (29 of 32) versus 68.2% (30 of 44); P = 0.026. Clinical outcomes were not significantly different between the fresh and the frozen-thawed sperm group: clinical pregnancy rates were 7 of 10 (70.0%) and 8 of 16 (50.0%); implanted embryos (per transferred embryo) were 8 of 23 (34.8%) and 8 of 29 (27.6%); delivery rates were 6 of 10 (60.0%) and 5 of 16 (31.3%). Sixteen babies were born, all of them are healthy with a normal karyotype, eight from the fresh sperm group and eight from the frozen-thawed sperm group.The small numbers available for study mean that only common problems can be excluded.This study provides further reassurance that KS men can father healthy children and that pre-implantation genetic diagnosis on embryos conceived with their sperm is not strongly indicated. However, until conclusive information is available, such couples should be offered extensive genetic counseling.No external funding was obtained for the present study. None of the authors has any conflict of interest to declare.Not applicable.