Mature oocyte dysmorphisms may be associated with progesterone levels, mitochondrial DNA content, and vitality in luteal granulosa cells.

PURPOSE: To identify whether follicular environment parameters are associated with mature oocyte quality, embryological and clinical outcomes. METHODS: This retrospective study examined 303 mature oocytes from 51 infertile women undergoing ICSI cycles between May 2018 and June 2021. Exclusion criteria consisted of advanced maternal age (> 36 years old), premature ovarian failure, obesity in women, or use of frozen gametes. Luteal granulosa cells (LGCs) were analyzed for mitochondrial DNA/genomic (g) DNA ratio and vitality. The relationships between hormone levels in the follicular fluid and oocyte features were assessed. Quantitative morphometric measurements of mature oocytes were assessed, and the association of LGC parameters and oocyte features on live birth rate after single embryo transfer was examined. RESULTS: Results indicated an inverse correlation between the mtDNA/gDNA ratio of LGCs and the size of polar body I (PBI). A 4.0% decrease in PBI size was observed with each one-unit increase in the ratio (p = 0.04). Furthermore, a 1% increase in LGC vitality was linked to a 1.3% decrease in fragmented PBI (p = 0.03), and a 1 ng/mL increase in progesterone levels was associated with a 0.1% rise in oocytes with small inclusions (p = 0.015). Associations were drawn among LGC characteristics, perivitelline space (PVS) debris, cytoplasmic inclusions, PBI integrity, and progesterone levels. Certain dysmorphisms in mature oocytes were associated with embryo morphokinetics; however, live birth rates were not associated with follicular parameters and oocyte quality characteristics. CONCLUSION: Follicular markers may be associated with mature oocyte quality features.

Identifying predictors of Day 5 blastocyst utilization rate using an artificial neural network.

RESEARCH QUESTION: Can artificial intelligence identify predictors of an increased Day 5 blastocyst utilization rate (D5BUR), which is one of the most informative key performance indicators in an IVF laboratory? DESIGN: This retrospective, multicentre study evaluated six variables for predicting D5BUR using an artificial neural network (ANN): number of metaphase II (MII) oocytes injected (intracytoplasmic sperm injection); use of autologous/donated gametes; maternal age at oocyte retrieval; sperm concentration; progressive sperm motility rate; and fertilization rate. Cycles were divided into training and testing sets through stratified random sampling. D5BUR on Day 5 was grouped into <60% and ≥60% as per the Vienna consensus benchmark values. RESULTS: The area under the receiver operating characteristic curve (AUC) to predict the D5BUR groups was 80.2%. From the ANN model, all six independent variables were found to be of significant value for the prediction of D5BUR (P<0.0001), with the most important variable being the number of MII oocytes injected. Investigation of the effect of MII oocytes injected on D5BUR indicated an inverse correlation, with injection of an increasing number of MII oocytes resulting in a decreasing D5BUR (r=-0.344, P<0.001) and injection of up to six oocytes resulting in D5BUR ≥60%. CONCLUSION: The number of MII oocytes injected is the most important predictor of D5BUR. Exploration of additional variables and further validation of models that can predict D5BUR can guide the way towards personalized treatment and increased safety.

Lactobacillus plantarum secretions may exert a cryoprotective effect on human sperm motility: A prospective in vitro study.

BACKGROUND: Semen cryopreservation is a widely used procedure for fertility preservation, despite some level of cryodamage that may occur in spermatozoa after thawing. However, there is some evidence that lactobacilli, one of the bacteria found in semen, might benefit sperm quality. OBJECTIVES: This study aims to determine whether the addition of Lactobacillus plantarum secretions to sperm freezing medium has an impact on sperm motility, morphology, and DNA fragmentation. MATERIALS AND METHODS: This is a prospective auto-controlled study. It was conducted on 30 raw semen samples from 30 infertile men attending a fertility center for semen analysis. Before freezing, all the samples were analyzed for motility, morphology, and DNA fragmentation percentages. Each sample was then divided equally into three aliquots. Cryopreservation was performed on each aliquot using one of the following three media: without Lactobacillus plantarum secretions (control group) or with 107 or 108 colony-forming units/mL Lactobacillus plantarum secretions. Sperm motility, morphology, and DNA integrity were evaluated after the cryopreservation media were added and after semen thawing. RESULTS: The results of this study indicated that after thawing, no statistically significant decrease in progressive motility and non-progressive percentages were detected in the sperm freezing medium supplemented with 108 colony-forming units/mL Lactobacillus plantarum secretions than the fresh raw semen. Moreover, multivariate linear regression model analyses showed that the progressive motility (p = 0.02), non-progressive motility (p = 0.016), and non-motile spermatozoa (p = 0.012) percentages were significantly decreased in the freezing medium (without Lactobacillus plantarum secretions) compared to the fresh raw semen. DISCUSSION AND CONCLUSION: To the best of our knowledge, this is the first study showing that Lactobacillus plantarum secretions had a cryoprotective effect on sperm motility when added to the sperm freezing medium. Furthermore, Lactobacillus plantarum secretions were found to protect sperm DNA integrity more effectively than the freezing medium without Lactobacillus plantarum secretions in non-normozoospermia group. Cryopreservation procedures must therefore be optimized to minimize any iatrogenically induced sperm DNA damage, given the correlation between sperm DNA damage and increased mutation loads in progeny.

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.

A systematic review and evidence assessment of monogenic gene-disease relationships in human female infertility and differences in sex development.

BACKGROUND: As in other domains of medicine, high-throughput sequencing methods have led to the identification of an ever-increasing number of gene variants in the fields of both male and female infertility. The increasing number of recently identified genes allows an accurate diagnosis for previously idiopathic cases of female infertility and more appropriate patient care. However, robust evidence of the gene-disease relationships (GDR) allowing the proper translation to clinical application is still missing in many cases. OBJECTIVE AND RATIONALE: An evidence-based curation of currently identified genes involved in female infertility and differences in sex development (DSD) would significantly improve both diagnostic performance and genetic research. We therefore performed a systematic review to summarize current knowledge and assess the available GDR. SEARCH METHODS: PRISMA guidelines were applied to curate all available information from PubMed and Web of Science on genetics of human female infertility and DSD leading to infertility, from 1 January 1988 to 1 November 2021. The reviewed pathologies include non-syndromic as well as syndromic female infertility, and endocrine and reproductive system disorders. The evidence that an identified phenotype is caused by pathogenic variants in a specific gene was assessed according to a standardized scoring system. A final score (no evidence, limited, moderate, strong, or definitive) was assigned to every GDR. OUTCOMES: A total of 45 271 publications were identified and screened for inclusion of which 1078 were selected for gene and variant extraction. We have identified 395 genes and validated 466 GDRs covering all reported monogenic causes of female infertility and DSD. Furthermore, we present a genetic diagnostic flowchart including 105 genes with at least moderate evidence for female infertility and suggest recommendations for future research. The study did not take into account associated genetic risk factor(s) or oligogenic/polygenic causes of female infertility. WIDER IMPLICATIONS: We have comprehensively reviewed the existing research on the genetics of female infertility and DSD, which will enable the development of diagnostic panels using validated genes. Whole genome analysis is shifting from predominantly research to clinical application, increasing its diagnostic potential. These new diagnostic possibilities will not only decrease the number of idiopathic cases but will also render genetic counselling more effective for infertile patients and their families.

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.

Functional histology of human scrotal wall layers and their overlooked relation with infertility: a narrative review.

Male infertility currently contributes to nearly half of the reported infertility cases. Scrotal wall layers play a cardinal role in regulating testicular physiology. However, few studies have focused on the functional histology of these layers and their relations with infertility in humans. The objective of the present narrative review is to collate novel insights into the functional histology of the human scrotal wall layers and their relation with infertility. The data was extracted from articles published between 1946 and 2021. The study was performed between January and December 2021. 71 original studies have been included in this review. Despite the fact that few studies have presented detailed functional histology of the human scrotal wall layers, this narrative review elucidates the possible influence of scrotal histology on infertility. Scrotal wall layers-associated pathologies may induce infertility by various mechanisms. They can impose mechanical forces that may affect the testicular histology and stimulate testicular inflammation. Moreover, they may induce testicular hyperthermia. Various unanswered clinical questions have been identified in this narrative review. More clinical studies are needed to assess the effect of alterations in the components of the scrotal wall layers on fertility (e.g., due to the exposure to metabolic and/or psychological stressors). In addition, testing the effectiveness of various pharmacological/surgical interventions to treat scrotal wall layers-associated pathologies will provide more insights into infertility treatment.