Mosaic human preimplantation embryos and their developmental potential in a prospective, non-selection clinical trial.

Chromosome imbalance (aneuploidy) is the major cause of pregnancy loss and congenital disorders in humans. Analyses of small biopsies from human embryos suggest that aneuploidy commonly originates during early divisions, resulting in mosaicism. However, the developmental potential of mosaic embryos remains unclear. We followed the distribution of aneuploid chromosomes across 73 unselected preimplantation embryos and 365 biopsies, sampled from four multifocal trophectoderm (TE) samples and the inner cell mass (ICM). When mosaicism impacted fewer than 50% of cells in one TE biopsy (low-medium mosaicism), only 1% of aneuploidies affected other portions of the embryo. A double-blinded prospective non-selection trial (NCT03673592) showed equivalent live-birth rates and miscarriage rates across 484 euploid, 282 low-grade mosaic, and 131 medium-grade mosaic embryos. No instances of mosaicism or uniparental disomy were detected in the ensuing pregnancies or newborns, and obstetrical and neonatal outcomes were similar between the study groups. Thus, low-medium mosaicism in the trophectoderm mostly arises after TE and ICM differentiation, and such embryos have equivalent developmental potential as fully euploid ones.

Incidence, Origin, and Predictive Model for the Detection and Clinical Management of Segmental Aneuploidies in Human Embryos.

Despite next-generation sequencing, which now allows for the accurate detection of segmental aneuploidies from in vitro fertilization embryo biopsies, the origin and characteristics of these aneuploidies are still relatively unknown. Using a multifocal biopsy approach (four trophectoderms [TEs] and one inner cell mass [ICM] analyzed per blastocyst; n = 390), we determine the origin of the aneuploidy and the diagnostic predictive value of segmental aneuploidy detection in TE biopsies toward the ICM's chromosomal constitution. Contrary to the prevalent meiotic origin of whole-chromosome aneuploidies, we show that sub-chromosomal abnormalities in human blastocysts arise from mitotic errors in around 70% of cases. As a consequence, the positive-predictive value toward ICM configuration was significantly lower for segmental as compared to whole-chromosome aneuploidies (70.8% versus 97.18%, respectively). In order to enhance the clinical utility of reporting segmental findings in clinical TE biopsies, we have developed and clinically verified a risk stratification model based on a second TE biopsy confirmation and segmental length; this model can significantly improve the prediction of aneuploidy risk in the ICM in over 86% of clinical cases enrolled. In conclusion, we provide evidence of the predominant mitotic origin of segmental aneuploidies in preimplantation embryos and develop a risk stratification model that can help post-test genetic counseling and that facilitates the decision-making process on clinical utilization of these embryos.

The use of copy number loads to designate mosaicism in blastocyst stage PGT-A cycles: fewer is better.

STUDY QUESTION: How well can whole chromosome copy number analysis from a single trophectoderm (TE) biopsy predict true mosaicism configurations in human blastocysts? SUMMARY ANSWER: When a single TE biopsy is tested, wide mosaicism thresholds (i.e. 20-80% of aneuploid cells) increase false positive calls compared to more stringent ones (i.e. 30-70% of aneuploid cells) without improving true detection rate, while binary classification (aneuploid/euploid) provides the highest diagnostic accuracy. WHAT IS KNOWN ALREADY: Next-generation sequencing-based technologies for preimplantation genetic testing for aneuploidies (PGT-A) allow the identification of intermediate chromosome copy number alterations potentially associated with chromosomal mosaicism in TE biopsies. Most validation studies are based on models mimicking mosaicism, e.g. mixtures of cell lines, and cannot be applied to the clinical interpretation of TE biopsy specimens. STUDY DESIGN, SIZE, DURATION: The accuracy of different mosaicism diagnostic thresholds was assessed by comparing chromosome copy numbers in multiple samples from each blastocyst. Enrolled embryos were donated for research between June 2019 and September 2020. The Institutional Review Board at the Near East University approved the study (project: YDU/2019/70-849). Embryos showing euploid/aneuploid mosaicism (n = 53), uniform chromosomal alterations (single or multiple) (n = 25), or uniform euploidy (n = 39) in their clinical TE biopsy were disaggregated into five portions: the inner cell mass (ICM) and four TE segments. Collectively, 585 samples from 117 embryos were analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated blastocysts were warmed, allowed to re-expand, and disaggregated in TE portions and ICM. PGT-A analysis was performed using Ion ReproSeq PGS kit and Ion S5 sequencer (ThermoFisher). Sequencing data were blindly analysed with Ion Reporter software to estimate raw chromosome copy numbers. Intra-blastocyst comparison of copy number data was performed employing different thresholds commonly used for mosaicism detection. From copy number data, different case scenarios were created using more stringent (30-70%) or less stringent criteria (20-80%). Categorical variables were compared using the two-sample z test for proportions. MAIN RESULTS AND THE ROLE OF CHANCE: When all the five biopsies from the same embryo were analysed with 30-70% thresholds, only 8.4% (n = 14/166) of patterns abnormal in the original analysis revealed a true mosaic configuration, displaying evidence of reciprocal events (3.6%, n = 6/166) or confirmation in additional biopsies (4.8%, n = 8/166), while most mosaic results (87.3% of total predicted mosaic patterns) remained confined to a single TE specimen. Conversely, uniform whole chromosome aneuploidies (28.3% of total patterns, n = 47/166) were confirmed in all subsequent biopsies in 97.9% of cases (n = 46/47). When 20-80% thresholds were employed (instead of 30-70%), the overall mosaicism rate per biopsy increased from 20.2% (n = 114/565) to 40.2% (n = 227/565). However, the use of a wider threshold range did not contribute to the detection of additional true mosaic patterns, while significantly increasing false positive mosaic patterns from 57.8% to 79.5% (n = 96/166; 95% CI = 49.9-65.4 vs n = 271/341; 95% CI = 74.8-83.6, respectively) (P < 0.00001). Moreover, the shift of the aneuploid cut-off from 70% to 80% of aneuploid cells resulted in mosaicism overcalling in the high range (50-80% of aneuploid cells), impacting the accuracy of uniform aneuploid classification. Parametric analysis of thresholds, based on multifocal analysis, revealed that a binary classification scheme with a single cut-off at a 50% level provided the highest sensitivity and specificity rates. Further analysis on technical noise distribution at the chromosome level revealed a greater impact on smaller chromosomes. LIMITATIONS, REASONS FOR CAUTION: While enrolment of a population enriched in embryos showing intermediate chromosome copy numbers enhanced the evaluation of the mosaicism category compared with random sampling such study population selection is likely to lead to an overall underestimation of PGT-A accuracy compared to a general assessment of unselected clinical samples. This approach involved the analysis of aneuploidy chromosome copy number thresholds at the embryo level; future studies will need to evaluate these criteria in relation to clinical predictive values following embryo transfers for different PGT-A assays. Moreover, the study lacked genotyping-based confirmation analysis. Finally, aneuploid embryos with known meiotic partial deletion/duplication were not included. WIDER IMPLICATIONS OF THE FINDINGS: Current technologies can detect low-intermediate chromosome copy numbers in preimplantation embryos but their identification is poorly correlated with consistent propagation of the anomaly throughout the embryo or with negative clinical consequences when transferred. Therefore, when a single TE biopsy is analysed, diagnosis of chromosomal mosaicism should be evaluated carefully. Indeed, the use of wider mosaicism thresholds (i.e. 20-80%) should be avoided as it reduces the overall PGT-A diagnostic accuracy by increasing the risk of false positive mosaic classification and false negative aneuploid classification. From a clinical perspective, this approach has negative consequences for patients as it leads to the potential deselection of normal embryos for transfer. Moreover, a proportion of uniform aneuploid embryos may be inaccurately categorized as high-level mosaic, with a consequent negative outcome (i.e. miscarriage) when inadvertently selected for transfer. Clinical outcomes following PGT-A are maximized when a 50% threshold is employed as it offers the most accurate diagnostic approach. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by Igenomix. The authors not employed by Igenomix have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Fertilization and development of oocytes with separated and conjoined zona pellucida recovered from polyovular follicles: description of two cases and a literature review.

Recovery of more than one oocyte from a single follicle during laparoscopic egg collection has been reported sporadically and accepted as confirmation of the presence of polyovular or binovular follicles in the human ovary at reproductive age. Most of these reports include conjoined oocytes that share common or fused zona pellucida, and are generally accepted as evidence for true polyovularity due to its certain characteristics. In this study, we report one case of a conjoined oocyte and another case of the recovery of two separate oocytes in a cumulus cell complex and details of their early embryonic development. To our knowledge, this report of the recovery of two separate oocytes without zonal contact is the first in the literature. We reviewed the relevant literature to evaluate information regarding the origin, incidence and significance of polyovularity in reproductive health.

5-Alfa reductase type 2 (SRD5A2) gene rs523349 polymorphism is not associated with non-obstructive azoospermia in Turkish patients.

Spermatogenesis is an androgen-dependent event, and testosterone is the major androgen source. The enzyme 5-alpha reductase converts testosterone to dihydrotestosterone (DHT) in testicular and peripheral tissues. Polymorphisms in genes encoding 5-alpha reductase may be associated with impaired male fertility. The present study aimed to investigate the relationship between 5-alpha reductase type 2 (SRD5A2) gene rs523349 polymorphism and non-obstructive azoospermia (NOA) in Turkish patients. The study included 75 NOA patients and 43 fertile men from Turkey. No significant relationship was found between SRD5A2 gene rs523349 polymorphism and male infertility (P = 0.071). There was a statistically significant difference in total testosterone level and total testis volume between NOA patients and the control groups, however there was no significant difference between serum follicle-stimulating hormone and luteinizing hormone levels. Our results showed that SRD5A2 gene rs523349 polymorphism was not associated with NOA in Turkish patients.

Measuring the serum progesterone level on the day of transfer can be an additional tool to maximize ongoing pregnancies in single euploid frozen blastocyst transfers.

BACKGROUND: Endometrial preparation with hormone replacement therapy (HRT) is the preferred regimen for clinicians due to the opportunity to schedule the day of embryo transfer and for patients due to the requirement of fewer visits for frozen-warmed embryo transfers (FET). The increasing number of FETs raises the question of the serum P levels required to optimize the pregnancy outcome on the embryo transfer day. METHODS: This prospective cohort study includes patients who underwent single euploid FET. All patients received HRT with oestradiol valerate (EV) and 100 mg of intramuscular (IM) progesterone (P). FET was scheduled 117-120 h after the first IM administration of 100 mg P. The serum P level was analyzed 1 h before the embryo transfer (ET). In all cycles, only embryos that were biopsied on day 5 were utilized for FET. Next generation sequencing (NGS) was used for comprehensive chromosomal analysis. RESULTS: Overall, the ongoing pregnancy rate (OPR) was 58.9% (99/168). Data were then categorized according to the presence (Group I; n = 99) or the absence (Group II; n = 69) of an ongoing pregnancy. No significant differences regarding, female age, body mass index (BMI), number of previous miscarriages, number of previous live birth, sperm concentration, number of oocytes retrieved, number of mature oocytes (MII), rate of fertilized oocytes with two pronuclei (2PN), trophectoderm score, inner cell mass (ICM) score, endometrial thickness (mm), oestrodiol (E2) and P levels prior to IM P administration were found between two groups. The P levels on the day of ET (ng/ml) were significantly higher in Group I (28 (5.6-76.4) vs 16.4 (7.4-60) p = 0.039). The P level on the day of ET was a predictor of a higher OPR (p < 0.001 OR: 1.033 95%CI [1.009-1.056]) after multivariate analysis. The ROC curve showed a significant predictive value of serum P levels on the day of ET for OPR, with an AUC (95%CI) = 0.716 (0.637-0.795). The optimal cut-off value for prediction of the OPR was a P level of 20.6 ng/ml (71.7% sensitivity, 56.5% specificity). CONCLUSIONS: The present study suggests a minimum threshold of the serum P value on the day of ET that needs to be reached in HRT cycles to optimize the clinical outcome. Individualization of the P dosage should be evaluated in further studies.

Assessment of Repetitive Controlled Ovarian Stimulation (COS) Cycles on Oocyte Donors: Impact on Oocyte Quality and Viable Embryo Yield.

The utilization of donor eggs has broadened the options for Assisted Reproductive Technology (ART) among women facing challenges with egg quantity or quality. Given that donors are typically selected from young and fertile individuals, In Vitro Fertilization with egg donation (IVF-ED) tends to exhibit higher rates of implantation, pregnancy, and live births compared to IVF with the woman's own eggs, especially for females over 35 years old. This has led to a projected increase in the demand for IVF-ED, surpassing the number of available donors. Consequently, many centers opt to use oocyte donors for multiple cycles. However, the correlation between repeated Controlled Ovarian Stimulation (COS) cycles and the performance of donors in terms of viable blastocyst stage embryo (VEC) or blastocyst embryo rate is not definitively established and remains of interest. This study aims to explore the preimplantation characteristics of embryo development and oocyte maturation status based on the number of donor COS cycles, employing a Generalized Linear Mixed Model (GLMM) framework. The study encompasses 1965 embryo transfer (ET) cycles involving 399 donors who underwent a minimum of two and a maximum of nine controlled ovarian hyperstimulation (COS) cycles. The findings indicate that, with the patient undergoing six or more cycles of ovarian stimulation, despite a 3.9% increase in both maturation and fertilization rates, there is a corresponding decrease of 4.5% in VEC rate and 4.7% in blastulation rates. In essence, an escalating number of donor COS cycles appears to be associated with a disadvantageous reduction in embryo quality.

Effect of paternal age on assisted reproductive outcomes in ICSI donor cycles.

BACKGROUND: Despite growing evidence suggesting age-related molecular changes in gametes, the impact of paternal age on clinical outcomes during infertility treatments has not been adequately assessed. OBJECTIVES: This study aims to assess the correlation of paternal age to clinical pregnancy and live birth rates in egg donation cycles undergoing intracytoplasmic sperm injection. MATERIALS AND METHODS: This retrospective cohort study includes 4930 fresh oocyte donation cycles from 3995 couples between April 2005 and February 2020 in a private IVF hospital. Clinical pregnancy and live birth rates were the primary outcome measures. The results were also assessed according to the paternal age groups, donor characteristics, semen parameters, fertilization rate, and quality of the transferred embryos. RESULTS: The age and body mass index of the donors, oocyte maturation, fertilization rates, and the mean number of transferred embryo quality were comparable on day-3 but not on day-5 embryo transfers between paternal age groups (p > 0.05). Paternal age was found to be negatively correlated to the number of oocytes utilized, normal semen parameters, fertilization, clinical pregnancy, and live birth rates (p < 0.05). In day-5 embryo transfer cycles, only the rate of cycles with normal spermatozoa, number of allocated oocytes, and pregnancy were found to be statistically significant. DISCUSSION AND CONCLUSION: Paternal age may influence reproductive outcomes and should be considered during infertility evaluations in intracytoplasmic sperm injection donor cycles. Further research is needed to confirm these findings.

Parameters impacting the live birth rate per transfer after frozen single euploid blastocyst transfer.

BACKGROUND: To assess the predictive value of patient characteristics, controlled ovarian stimulation and embryological parameters on the live birth outcome of single euploid frozen-warmed blastocyst transfer (FBT). METHODS: This was a retrospective cohort study including 707 single FBTs after preimplantation genetic testing for aneuploidy (PGT-A) that were performed from October 1, 2015, to January 1, 2018. The effects of patient-, cycle- and embryology-related parameters on the live birth outcome after FBT were assessed. RESULTS: In the subgroup analysis based on live birth, patients who achieved a live birth had a significantly lower body mass index (BMI) than patients who did not achieve a live birth (22.7 (21.5-24.6) kg/m2 vs 27 (24-29.2) kg/m2, p<0.001). The percentage of blastocysts with inner cell mass (ICM) A or B was significantly higher among patients achieving a live birth, at 91.6% vs. 82.6% (p<0.001). Day-5 biopsies were also more prevalent among patients achieving a live birth, at 82.9% vs 68.1% (p<0.001). On the other hand, the mitochondrial DNA (mtDNA) levels were significantly lower among cases with a successful live birth, at 18.7 (15.45-23.68) vs 20.55 (16.43-25.22) (p = 0.001). The logistic regression analysis showed that BMI (p<0.001, OR: 0.789, 95% CI [0.734-0.848]), day of trophectoderm (TE) biopsy (p<0.001, OR: 0.336, 95% CI [0.189-0.598]) and number of previous miscarriages (p = 0.004, OR: 0.733, 95% CI [0.594-0.906]) were significantly correlated with live birth. Patients with elevated BMIs, cycles in which embryos were biopsied on day-6 and a higher number of miscarriages were at increased risks of reduced live birth rates. CONCLUSION: A high BMI, an embryo biopsy on day-6 and a high number of miscarriages negatively affect the live birth rate after single euploid FBT.

Successful application of a single warming protocol for embryos cryopreserved by either slow freezing or vitrification techniques.

The aim of this study was to evaluate the feasibility and efficiency of using a sucrose gradient-based warming protocol as a universal warming approach on human cleavage stage embryos. Between January 2013 and November 2014, a total of 118 warming cycles were performed on 705 embryos which had previously been cryopreserved/thawed by slow freezing protocols or cryopreserved by slow freezing and warmed by vitrification thaw solution. Clinical outcomes have been retrospectively analyzed depending on cryopreservation and warming techniques used, embryo viability, day of cryopreservation, clinical pregnancy, implantation, and live birth rate. Results indicate that, the use of the vitrification warming protocol for warming after slow freezing results in comparable post-warming survival (71.6% and 71.1%; p = 0.890). Higher clinical pregnancy, implantation, and live birth rates were obtained in the cryopreserved embryos by slow freezing and warmed by vitrification group in comparison to the cryopreserved/thawed by slow freezing protocols group but the results did not show statistically significant differences between groups (p > 0.05). These results indicate that such an approach can eliminate the need to search for a brand-dependent product, as well as case-dependent hands-on planning. Further research that evaluates the effectiveness of this approach on a larger case series is underway. Abbreviations: CPA: concentrated cryoprotective agent; COH: controlled ovarian stimulation; FET: frozen embryo transfer; HSG: hysterosalpingogram; mHTF: modified human tubal medium; SSM: single step media; SSS: synthetic serum substitute; TV-USG: transvaginal ultrasound.

The impact of endometriosis on early embryo morphokinetics: a case-control study.

The aim of this study was to evaluate the possible effects of endometriosis on early embryo development, by comparing the morphokinetic development of embryos obtained from women with clinically confirmed endometriosis with the ones obtained from tubal factor infertility cases. A total of 82 cycles/patients including 53 cycles with endometriosis and 29 cycles with tubal factor infertility were evaluated. A total of 439 embryos were scored for embryo morphokinetics. Age, body mass index, fertilization rates were similar within the groups. However, the number of previous ART trials was found to be higher (p < 0.05) in the study group. Also, the number of retrieved oocytes and M2 oocytes were found to be significantly lower in patients with endometriosis (p < 0.01). The duration of the first cell cycle (ECC1) and S2 (the time between t3 and t4) displayed significant distortions compared with embryos in the control group. All other analyzed early morphokinetic parameters (t2, t3, t4, t5, t6, t7, t8) and duration of events (VP, cc2a, ECC2, ECC3, S3) showed similar values between study and control groups, respectively. In the light of these findings, it is apparent that endometriosis predominantly affects the duration of the early morphokinetic events and cell cycles.

Preliminary study of embryo development following assessment of male and female gametes.

Gamete characteristics are likely to contribute to embryo development. In this preliminary study, early parameters were assessed in embryos generated following microinjection of hyaluronan-bound and unbound spermatozoa into 176 sibling oocytes, which had themselves been assessed using a polarizing light microscope imaging system. The fertilization and early cleavage rates, and day 3 embryo quality did not differ when oocytes displaying similar characteristics were inseminated with either bound or unbound sperm. Regardless of the binding characteristics of the sperm, early embryos displaying good and poor quality derived from oocytes displaying visible spindles and similar characteristics. These results indicate that early embryo developmental characteristics were independent from the hyaluronic acid binding capacity of the sperm when oocytes displayed a visible spindle.

Evaluation of the impact of sperm morphology on embryo aneuploidy rates in a donor oocyte program.

This study investigates the correlation between sperm morphology and the incidence of embryo aneuploidy in an oocyte donation program. A total of 1,165 embryos from 103 patients have been analyzed by fluorescent in situ hybridization (FISH) for numerical abnormalities in chromosome numbers 13, 18, 21, X, and Y. Data has been evaluated in five groups according to sperm morphology, which has been assessed according to the Kruger's strict criteria. The results did not show any difference in paternal (p = 0.878), maternal (p = 0.873), and donor ages (p = 0.871), sperm counts (p = 0.782) and motility (p = 0.124), and fertilization rate (p = 0.080) among the groups. However, total aneuploidy rate (p < 0.001) and its derivatives (trisomy p = 0,042, monosomy p = 0,004) differed significantly and they were reversibly correlated with sperm morphology (rho correlation test; total aneuploidy p < 0.001, trisomy p < 0.001, monosomy p = 0.004). Therefore, these results suggested that diminished sperm quality is correlated to the aneuploidy rate in preimplantation embryos. ABBREVIATIONS: FISH: fluorescence in situ hybridization; ICSI: intracytoplasmic sperm injection; HCG: human chorionic gonadotropin.

Birth after 12 hours of oocyte refrigeration.

OBJECTIVE: To assess cycle outcome after oocyte refrigeration. DESIGN: Case report. SETTING: Private IVF center. PATIENT(S): One couple in a donor oocyte program. INTERVENTION(S): Intracytoplasmic sperm injection and blastocyst culture after refrigeration of oocytes for 12 hours. MAIN OUTCOME MEASURE(S): Birth. RESULT(S): Fourteen two-pronuclei zygotes from 17 metaphase II refrigerated oocytes resulted in transfer of two blastocysts at day 5 and cryopreservation of six excess embryos at day 6. The patient delivered one healthy male baby after 38 weeks' gestation. CONCLUSION(S): The successful outcome of oocyte refrigeration indicates that this protocol could be useful in circumstances in which a delay in obtaining spermatozoa arises.