Telomerase activity, telomere length, and the euploidy rate of human embryos.

BACKGROUND: Telomeres maintain chromosome stability, while telomerase counteracts their progressive shortening. Telomere length varies between cell types, with leukocyte telomere length (LTL) decreasing with age. Reduced telomerase activity has been linked to reproductive issues in females, such as low pregnancy rates and premature ovarian failure, with recent studies indicating correlations between telomere length in granulosa cells and IVF outcomes. OBJECTIVES: The study aims to explore the relationship between telomere length, telomerase activity, and euploid blastocyst rate in infertile women undergoing IVF/ICSI PGT-A cycles. METHODS: This prospective study involves 108 patients undergoing controlled ovarian stimulation and PGT-A. Telomere length and telomerase activity were measured in peripheral mononuclear cells and granulosa cells (GC), respectively. RESULTS: The telomere repeat copy number to single gene copy number ratio (T/S) results respectively 0.6 ± 0.8 in leukocytes and 0.7 ± 0.9 in GC. An inverse relationship was found between LTL and the patient's age (p < .01). A higher aneuploid rate was noticed in patients with short LTL, with no differences in ovarian reserve markers (p = .15), number of oocytes retrieved (p = .33), and number of MII (p = 0.42). No significant association was noticed between telomere length in GC and patients' age (p = 0.95), in ovarian reserve markers (p = 0.32), number of oocytes retrieved (p = .58), number of MII (p = .74) and aneuploidy rate (p = .65). CONCLUSION: LTL shows a significant inverse correlation with patient age and higher aneuploidy rates. Telomere length in GCs does not correlate with patient age or reproductive outcomes, indicating differential telomere dynamics between leukocytes and granulosa cells.

Two clinical case reports of embryonic mosaicism identified with PGT-A persisting during pregnancy as true fetal mosaicism.

The health risks associated with transferring embryos classified as mosaic by preimplantation genetic testing for aneuploidies (PGT-A) are currently unknown. Such embryos produce PGT-A results indicating the presence of both euploid and aneuploid cells and have historically been deselected from transfer and grouped with uniformly aneuploid embryos as 'abnormal'. In recent years, numerous groups have reported the intentional transfer of mosaic embryos in the absence of uniformly euploid embryos, largely observing births of seemingly healthy babies. However, it remains to be understood whether the embryonic mosaicism invariably becomes resolved during the ensuing pregnancy, or whether the placenta and/or fetal tissues retain aneuploid cells, and if so to what potential clinical effect. Here, we report two cases of mosaicism persisting from the embryonic stage to the established pregnancy. Case 1 involved an embryonic low-level segmental mosaic loss in Chromosome (Chr) 1, which was confirmed in amniocentesis as well as in brain tissue of the products of conception. This pregnancy was terminated due to the chromosomal pathologies associated with 1p36 deletion syndrome, such as severe intellectual disability. Case 2 involved a low-level mosaic Chr 21 trisomy, which was confirmed with chorionic villus sampling and amniocentesis. The ensuing pregnancy was terminated after ultrasound identification of severe abnormalities in the placenta and fetus. Together, these two cases should be taken into account for risk-benefit assessments of prospective mosaic embryo transfers.

PGT-M, a Useful Tool to Manage the Lynch Syndrome Transmission.

Lynch syndrome is one of the most common hereditary cancer sensitivity syndromes and is caused by autosomal-dominant germline mutations in DNA mismatch repair genes. In patients affected by this syndrome, pre-implantation genetic testing for monogenic disorders (PGT-M) could be the elective technique used to prevent the transmission of this hereditary syndrome to offspring. Notably, despite the severity of the condition, some authors have observed a markedly lower demand for PGT-M in these patients compared to those with other hereditary conditions. A 34-year-old woman with a medical history of Lynch syndrome associated with endometrial cancer came to the Villa Mafalda fertility center in Rome in order to conceive a healthy baby. In a pre-implantation genetic testing for aneuploidy (PGT-A) + PGT-M cycle, eight blastocysts were formed. Six out of eight blastocysts were affected by the same mother syndrome. One of the other two was aneuploid and the other one was a mosaic embryo, which resulted in a healthy pregnancy. The aim of this report is to emphasize the importance of a multidisciplinary approach to managing patients with this condition. In vitro fertilization (IVF), specifically PGT-M, is a tool that allow patients to conceive biological children with lower risk of inheriting the disease.

The number and rate of euploid blastocysts in women undergoing IVF/ICSI cycles are strongly dependent on ovarian reserve and female age.

STUDY QUESTION: Can the possibility of having at least one euploid blastocyst for embryo transfer and the total number of euploid blastocysts be predicted for couples before they enter the IVF programme? SUMMARY ANSWER: Ovarian reserve and female age are the most important predictors of having at least one euploid blastocyst and the total number of euploid blastocysts. WHAT IS KNOWN ALREADY: The blastocyst euploidy rate among women undergoing ART has already been shown to significantly decrease with increasing female age, and the total number of euploid embryos is dependent on the blastocyst cohort size. However, the vast majority of published studies are based on retrospective analysis of data. STUDY DESIGN, SIZE, DURATION: This prospective analysis included 847 consecutively enrolled couples approaching their first preimplantation genetic testing for aneuploidies (PGT-A) cycle between 2017 and 2020. Only couples for whom ejaculated sperm was available and women with a BMI of <35 kg/m2 were included in the study. Only the first cycle was included for each patient. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted at an IVF centre where, for all patients, the planned treatment was to obtain embryos at the blastocyst stage for the PGT-A programme. The impact of the following covariates was investigated: a woman's serum AMH level, age, height, weight and BMI and a man's age, height, weight, BMI, sperm volume and sperm motility and morphology. The analysis was performed with a machine learning (ML) approach. Models were fit on the training set (677 patients) and their predictive performance was then evaluated on the test set (170 patients). MAIN RESULTS AND ROLE OF CHANCE: After ovarian stimulation and oocyte insemination, 40.1% of couples had at least one blastocyst available for the PGT-A. Of 1068 blastocysts analysed, 33.6% were euploid. Two distinct ML models were fit: one for the probability of having at least one euploid blastocyst and one for the number of euploid blastocysts obtained. In the training set of patients, the variable importance plots of both models indicated that AMH and the woman's age are by far the most important predictors. Specifically, a positive association between the outcome and AMH and a negative association between the outcome and female age appeared. Gradient-boosted modelling offers a greater predictive performance than generalized additive models (GAMs). LIMITATIONS, REASONS FOR CAUTION: The study was performed based on data from a single centre. While this provides a robust set of data with a constant ART process and laboratory practice, the model might be suitable only for the evaluated population, which may limit the generalization of the model to other populations. WIDER IMPLICATIONS OF THE FINDINGS: ML models indicate that for couples entering the IVF/PGT-A programme, ovarian reserve, which is known to vary with age, is the most important predictor of having at least one euploid embryo. According to the GAM, the probability of a 30-year-old woman having at least one euploid embryo is 28% or 47% if her AMH level is 1 or 3 ng/ml, respectively; if the woman is 40 years old, this probability is 18% with an AMH of 1 ng/ml and 30% with an AMH of 3 ng/ml. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by an unrestricted grant from Gedeon Richter. The authors declared no conflict of interests. TRIAL REGISTRATION NUMBER: N/A.

Which factors influence the success rate of egg donation programmes with imported vitrified oocytes?

RESEARCH QUESTION: Which factors influence the success rate of egg donation programmes with imported vitrified oocytes? DESIGN: 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. RESULTS: 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. CONCLUSION: 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.

COVID-19 - compliant - IVF: reorganized clinical practice taking into account the pandemic.

PURPOSE: The need for protective measures during the COVID-19 pandemic forced assisted reproduction centers to adopt new protocols in order to reduce the risk of infection for patients and staff. A COVID-19 Compliant IVF (in vitro fertilization) program based on the use of follitropin delta to induce ovarian stimulation and fixed protocol with GnRH (Gonadotropin-Releasing Hormone) antagonists was applied to demonstrate the efficacy of this program in reducing the necessity of patients to be present within the clinic while maintaining the expected success rate. METHODS: The program was applied to 100 patients (women aged between 20 and 40 years, at their first IVF cycle): pretreatment assessment was carried out via video conference and clinical documentations were sent by email. Ovarian stimulation was based on doses of Follitropin delta selected on the basis of body weight and serum AMH (Anti-Müllerian Hormone). The study population was then compared with historical controls to assess the percentage of patients receiving fresh embryo transfer. Finally, a questionnaire was proposed to assess patient satisfaction. RESULTS: The ovarian stimulation protocol with a fixed dose of follitropin delta led to an optimal ovarian response (8-14 oocytes) in 54% of the women included in the study. The rate of hyper response was 15% and was significantly lower that what reported in the control group (26.8%, p < .05). The good control of ovarian response to stimulation is the reason for the large percentage of cycles associated with fresh embryo transfer (84%, n 84/100). The rate of fresh embryo transfer was significantly higher that what reported for the control population (68.5%, p < .05). In 40 out of 84 patients, blastocysts were available for the fresh embryo transfer procedure. Eighty-five percent (89/100) of survey responders reported being extremely satisfied with the simplified approach to the IVF cycle because of reduced number of visits, easy management of working hours and reduction in travel costs. CONCLUSIONS: The use of follitropin delta and fixed protocol with GnRH antagonist in COVID-19 - Compliant IVF program is associated to a low percentage of patients developing excessive ovarian response compared to conventional strategy and a very good level of patient satisfaction. Beyond the pandemic, the new approach to ovarian stimulation, modern technologies and telemedicine could play a key role in bridging the gap between different geographic areas and could offer advantages such as increased accessibility, cost saving and patient satisfaction.

Can sperm quality influence embryo development and its ploidy? Analysis of 811 blastocysts obtained from different sperm sources.

The 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.

Monozygotic twin rate among ART centers: a multicenter analysis of data from 18 Italian units.

PURPOSE: The risk of monozygotic twins (MZTs) is increased in couples undergoing assisted reproductive technology (ART) treatments. Several systematic reviews have investigated the possible determinants linked to ART, but results obtained have not been conclusive. The study aims to investigate whether the incidence of MZT differed among ART centers. METHODS: This is a multicenter retrospective cohort study using the Italian ART National Registry database and involving the centers reporting data from individual ART cycles from 2015 to 2019. To investigate the incidence of MZT, only single embryo transfer cycles were considered. Women who had sex-discordant deliveries were excluded. MZT rate was calculated as the number of multiple pregnancies (more than one gestational sac at first ultrasound) out of the total number of clinical pregnancies. A binomial distribution model was used to determine the 95% CI of the frequency of MZT. RESULTS: Eighteen centers were included, and they provided data on 10,433 pregnancies. The total number of MZT was 162, corresponding to an incidence of 1.5% (95% CI: 1.3-1.8%). The rate of MZT among centers varied between 0% (95% CI: 0.0-25.9%) and 3.2% (95% CI: 1.3-8.1%). All the 95% CIs included 1.5%, rejecting the hypothesis that the MZT rate may significantly differ among centers. CONCLUSIONS: The rate of MZT did not significantly vary among ART centers. Local factors are unlikely to explain the increased rate of MZT in ART pregnancies.

Comparison of euploidy rates of blastocysts in women treated with progestins or GnRH antagonist to prevent the luteinizing hormone surge during ovarian stimulation.

STUDY QUESTION: Does the prevalence of euploid blastocysts differ between patients treated with progestin primed ovarian stimulation (PPOS) and those treated with conventional ovarian stimulation? SUMMARY ANSWER: The numbers of blastocysts and euploid blastocysts per patient and the number of euploid embryos per injected oocyte are similar for patients undergoing progestin-primed ovarian stimulation and for those undergoing conventional ovarian stimulation with GnRH antagonist. WHAT IS KNOWN ALREADY: New approaches to ovarian stimulation have been developed based on the use of drugs administrable by mouth instead of via injections. Attention has been dedicated to progestins to block the LH surge. Previous data regarding the number of oocytes retrieved and the number of good-quality embryos generated in PPOS have demonstrated similar outcomes when compared to conventional ovarian stimulation, even if some concerns regarding the quality of embryos have been advanced. STUDY DESIGN, SIZE, DURATION: This is a prospective non-inferiority age-matched case-control study. In a period of 6 months, a total of 785 blastocysts from 1867 injected oocytes obtained from 192 patients were available for analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile women undergoing IVF and preimplanation genetic testing (PGT) cycles were included. Forty-eight patients were treated with PPOS, and for each of them three age-matched historical controls (n = 144) treated with a GnRH antagonist protocol were selected. PGT was performed according to next-generation sequencing technology. MAIN RESULTS AND THE ROLE OF CHANCE: Basal characteristics were similar in the two groups; a substantial similarity of the main outcome measures in the two treatment groups has also been found. The rate of formation of euploid blastocysts per oocyte was 21% in both the two treatment groups. The percentage of patients with euploid embryos and the total number of euploid blastocysts per patient (median and interquartile range, IQR) in the PPOS group were 38.7 (25.5-52.9) and 2 (1.3-3.1), respectively. These figures were not significantly different in women treated with the GnRH antagonist protocol i.e. 42 (28-53.8) and 2.1 (1.3-2.9), respectively. LIMITATIONS, REASONS FOR CAUTION: This was a case-control study which may limit the reliability of the main findings. WIDER IMPLICATIONS OF THE FINDINGS: Our results encourage the use of PPOS, especially for oocyte donation, for fertility preservation and for patients in which total freezing of embryos is foreseen, for those expected to be high responders or candidates for preimplantation genetic testing. However, studies aiming to investigate the effect of PPOS on the live birth rate are warranted. STUDY FUNDING/COMPETING INTEREST(S): None.

Late Effects of Parasellar Lesion Treatment: Hypogonadism and Infertility.

Central hypogonadism, also defined as hypogonadotropic hypogonadism, is a recognized complication of hypothalamic-pituitary-gonadal axis damage following treatment of sellar and parasellar masses. In addition to radiotherapy and surgery, CTLA4-blocking antibodies and alkylating agents such as temozolomide can also lead to hypogonadism, through different mechanisms. Central hypogonadism in boys and girls may lead to pubertal delay or arrest, impairing full development of the genitalia and secondary sexual characteristics. Alternatively, cranial irradiation or ectopic hormone production may instead cause early puberty, affecting hypothalamic control of the gonadostat. Given the reproductive risks, discussion of fertility preservation options and referral to reproductive specialists before treatment is essential. Steroid hormone replacement can interfere with other replacement therapies and may require specific dose adjustments. Adequate gonadotropin stimulation therapy may enable patients to restore gametogenesis and conceive spontaneously. When assisted reproductive technology is needed, protocols must be tailored to account for possible long-term gonadotropin insufficiency prior to stimulation. The aim of this review was to provide an overview of the risk factors for hypogonadism and infertility in patients treated for parasellar lesions and to give a summary of the current recommendations for management and follow-up of these dysfunctions in such patients. We have also briefly summarized evidence on the physiological role of pituitary hormones during pregnancy, focusing on the management of pituitary deficiencies.

Preimplantation Genetic Testing: Where We Are Today.

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.

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.

Dynamic contrast-enhanced and diffusion-weighted MR imaging in the characterisation of small, non-palpable solid testicular tumours.

OBJECTIVES: To explore the role of dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI), using semiquantitative and quantitative parameters, and diffusion-weighted (DW) MRI in differentiating benign from malignant small, non-palpable solid testicular tumours. METHODS: We calculated the following DCE-MRI parameters of 47 small, non-palpable solid testicular tumours: peak enhancement (PE), time to peak (TTP), percentage of peak enhancement (Epeak), wash-in-rate (WIR), signal enhancement ratio (SER), volume transfer constant (Ktrans), rate constant (Kep), extravascular extracellular space volume fraction (Ve) and initial area under the curve (iAUC). DWI signal intensity and apparent diffusion coefficient (ADC) values were evaluated. RESULTS: Epeak, WIR, Ktrans , Kep and iAUC were higher and TTP shorter in benign compared to malignant lesions (p < 0.05). All tumours had similar ADC values (p > 0.07). Subgroup analysis limited to the most frequent histologies - Leydig cell tumours (LCTs) and seminomas - replicated the findings of the entire set. Best diagnostic cutoff value for identification of seminomas: Ktrans ≤0.135 min-1, Kep ≤0.45 min-1, iAUC ≤10.96, WIR ≤1.11, Epeak ≤96.72, TTP >99 s. CONCLUSIONS: DCE-MRI parameters are valuable in differentiating between benign and malignant small, non-palpable testicular tumours, especially when characterising LCTs and seminomas. KEY POINTS: • DCE-MRI may be used to differentiate benign from malignant non-palpable testicular tumours. • Seminomas show lower Ktrans, Kep and iAUC values. • ADC values are not valuable in differentiating seminomas from LCTs. • Semiquantitative DCE-MRI may be used to characterise small, solid testicular tumours.

Testicular histopathology, semen analysis and FSH, predictive value of sperm retrieval: supportive counseling in case of reoperation after testicular sperm extraction (TESE).

BACKGROUND: To provide indicators for the likelihood of sperm retrieval in patients undergoing testicular sperm extraction is a major issue in the management of male infertility by TESE. The aim of our study was to determine the impact of different parameters, including testicular histopathology, on sperm retrieval in case of reoperation in patients undergoing testicular sperm extraction. METHODS: We retrospectively analyzed 486 patients who underwent sperm extraction for intracytoplasmic sperm injection and testicular biopsy. Histology was classified into: normal spermatogenesis; hypospermatogenesis (reduction in the number of normal spermatogenetic cells); maturation arrest (absence of the later stages of spermatogenesis); and Sertoli cell only (absence of germ cells). Semen analysis and serum FSH, LH and testosterone were measured. RESULTS: Four hundred thirty patients had non obstructive azoospermia, 53 severe oligozoospermia and 3 necrozoospermia. There were 307 (63%) successful sperm retrieval. Higher testicular volume, lower levels of FSH, and better histological features were predictive for sperm retrieval. The same parameters and younger age were predictive factors for shorter time for sperm recovery. After multivariable analysis, younger age, better semen parameters, better histological features and lower values of FSH remained predictive for shorter time for sperm retrieval while better semen and histology remained predictive factors for successful sperm retrieval. The predictive capacity of a score obtained by summing the points assigned for selected predictors (1 point for Sertoli cell only, 0.33 points for azoospermia, 0.004 points for each FSH mIU/ml) gave an area under the ROC curve of 0.843. CONCLUSIONS: This model can help the practitioner with counseling infertile men by reliably predicting the chance of obtaining spermatozoa with testicular sperm extraction when a repeat attempt is planned.

Influence of human sperm origin, testicular or ejaculated, on embryo morphokinetic development.

In this retrospective observational study (October 2014 - July 2016), the impact of sperm origin on embryo morphokinetics and on clinical outcomes after intracytoplasmic sperm injection was evaluated. The developmental kinetics of embryos obtained either with testicular sperm (40 cycles; testicular sperm group) or with thawed donor sperm (26 cycles; donor sperm group) was analysed up to day-3 of culture with a time-lapse incubation system. In the testicular sperm group, all patients were affected by nonobstructive azoospermia. The timing of second polar body extrusion (IIPB), and the time to reach the 4-cells (t4) and 9-cells (t9) stages, differed significantly between the two groups: the IIPB extrusion and t4 were anticipated, whereas t9 was retarded in the testicular sperm group. We hypothesise that a different sperm maturation grade may influence the timing of embryo development: an early paternal effect of testicular sperm could be manifested as an anticipation in the IIPB extrusion and in the time for reaching the 4-cells stage. Conversely, a later paternal effect could be visible as a retardation in the timing at which the embryo reaches the 9-cells stage. Interestingly, clinical outcomes did not differ between the two groups except the implantation rate which was significantly increased in the donor sperm group.

HCG administration after endogenous LH rise negatively influences pregnancy rate in modified natural cycle for frozen-thawed euploid blastocyst transfer: a pilot study.

PURPOSE: The aim of the present study was to evaluate whether in a modified natural cycle (modified-NC) for a frozen-thawed single euploid blastocyst transfer, a critical LH value, above which human chorionic gonadotropin (hCG) administration should be avoided, may be defined. METHODS: One hundred and sixty-seven patients underwent modified natural cycle in order to transfer a single frozen-thawed euploid blastocyst. All embryos were obtained by intracytoplasmic sperm injection and were biopsied at the blastocyst stage and analyzed by means of array comparative genomic hybridization (aCGH). Ovulation was induced using 10.000 IU hCG when the mean follicle diameter was at least of 17 mm, independently from LH values. The primary end points were the hCG-positive test and clinical pregnancy. The interim analysis showed that LH value ≥ 13 mIU/ml on the day of hCG injection may negatively influence the clinical results, suggesting that in this condition, it should be advisable waiting for spontaneous ovulation. RESULTS: Among patients who received hCG for ovulation induction, the hCG-positive test and clinical pregnancy rates in modified-NC were significantly lower in cycles with LH ≥ 13 mIU/ml in respect to those with LH < 13 mIU/ml (45.4 vs 73.3 and 36.4 vs 65.9%, in LH ≥ 13 and LH < 13 groups, respectively). In patients with LH value ≥ 13 mIU/ml, hCG administration led to significantly lower rates of hCG-positive test (45.4 vs 74.5% in hCG administration and spontaneous ovulation groups, respectively) and clinical pregnancy (36.4 vs 64.7% in hCG administration and spontaneous ovulation groups, respectively). The baseline patient characteristics were comparable in all groups. CONCLUSIONS: The findings of this study highlight that LH elevation ≥ 13 mIU/ml prior to hCG administration may negatively affect clinical pregnancy rates in modified-NC for single euploid blastocyst transfer. The LH determination should be routinely performed during follicular monitoring. In the presence of LH level ≥ 13 mIU/ml, hCG administration should be avoided, and the embryo transfer should be planned only after spontaneous follicular rupture.

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.