Willem Verpoest

STUDY QUESTION: Which clinical definition of oocyte, zygote, and embryo maturation arrest (OZEMA) merits genetic investigation? SUMMARY ANSWER: The diagnosis OZEMA is considered when: only two mature oocytes are obtained from six cumulus–oocyte complexes (COCs), only one zygote is obtained from six metaphase II oocytes (MIIs), or no blastocysts are obtained from six two-pronuclei zygotes (2PNs). WHAT IS KNOWN ALREADY: OZEMA is a cover term for various defects, including oocyte abnormalities, fertilization failure, cleavage arrest, and abnormal embryo development, often associated with specific genetic variants. While some variants in specific genes are associated with distinct phenotypes, others commonly display variable expressivity across cycles and patients. Currently, there are no standardized cut-off values defining the OZEMA disorder. STUDY DESIGN, SIZE, DURATION: This is a pooled secondary analysis of aggregated data extracted from published studies. A PubMed search was conducted to identify peer-reviewed original studies in the human reporting genetically confirmed cases of OZEMA. Studies were included if patients had displayed OZEMA in at least two medically assisted reproduction (MAR) cycles. In total, clinical and genetic data were extracted from 132 publications, including 521 patients and 39 genes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Clinical phenotypes from 132 publications were classified into oocyte maturation arrest (OMA), zona pellucida abnormality (ZPA), fertilization failure (FF), zygote arrest (ZA), early embryo arrest (EEA), mixed phenotypes, and other abnormalities. Data at both cycle and patient levels were evaluated using ratios corresponding to key developmental milestones (ratio of MII/COC, 2PN/MII, cleaved embryos/2PN, blastocysts/2PN). These ratios were compared with the expected values Vienna Consensus binomial modeling to assess whether the observed proportions were significantly lower than the established competency thresholds (P < 0.05). MAIN RESULTS AND THE ROLE OF CHANCE: This review identified 39 OZEMA-associated genes. Analysis of 668 cycles from 253 patients carrying variants in 35 genes showed abnormal developmental outcomes in 93%, based on binomial modeling. Reliable diagnosis of OZEMA requires a sufficient number of COCs, MIIs, or 2PNs to distinguish true developmental impairment from normal biological variation. When these minimal thresholds are not reached, statistical power is insufficient to reliably confirm developmental arrest. Based on our findings, OZEMA is diagnosed when only two or fewer mature oocytes form from six COCs, one or no zygote forms from six MIIs, or no blastocysts form from six 2PNs. LIMITATIONS, REASONS FOR CAUTION: This study is limited by its retrospective design and reliance exclusively on published data from women selected for poor IVF outcomes and the presence of variants in candidate OZEMA genes, which may introduce publication bias and reporting heterogeneity. Therefore, this study does not report on genetic yield from women with normal IVF outcomes. Variability in clinical protocols and genetic testing methods, limited ancestral diversity, and missing data across studies could contribute to confounding factors. WIDER IMPLICATIONS OF THE FINDINGS: This study is an important step toward understanding the clinical and genetic spectrum of OZEMA, highlighting the variability of and overlap between affected subphenotypes. Establishing cut-off values against consensus oocyte and embryo developmental competency levels provides a benchmark for more accurate classification. Importantly, this represents the most comprehensive analysis of published OZEMA cases with genetic variants to date, strengthening the reliability of the proposed clinical thresholds. These findings may guide personalized treatment strategies and genetic counseling, ultimately improving outcomes in MAR. STUDY FUNDING/COMPETING INTEREST(S): No funding was received for this study. The authors declare no competing interests. REGISTRATION NUMBER: Not applicable.

Purpose Primary ovarian insufficiency (POI) is an important cause of female infertility, stemming from follicle dysfunction or premature oocyte depletion. Pathogenic variants in genes such as NOBOX, GDF9, BMP15, and FSHR have been linked to POI. NOBOX, a transcription factor expressed in oocytes and granulosa cells, plays a pivotal role in folliculogenesis. Loss-of-function variants in NOBOX are reported in 1–2% of POI women. This study aims to describe the association of novel NOBOX variants with a distinct oocyte, zygote, and embryo maturation arrest (OZEMA) phenotype in infertile women. Methods Three unrelated women experiencing OZEMA and undergoing multiple in vitro fertilization (IVF) cycles present with a germline NOBOX variant. The detected variants were cross-referenced with a large genetic database to explore their association with IVF outcomes. Results A heterozygous NM_001080413.3 (NOBOX): c.1797_1798del, p.(Cys600Phefs*27) variant was detected in a woman with oocyte maturation arrest. Another heterozygous variant, NM_001080413.3 (NOBOX): c.1849C > T, p.(His617Tyr), was detected in two women experiencing embryonic developmental arrest. Segregation analysis in one of the two latter families revealed the presence of the p.(His617Tyr) variant in an affected sister, while the two fertile sisters did not carry this variant. Furthermore, the p.(His617Tyr) variant was found in three women in a large database of whom two presented with an embryonic developmental arrest. Conclusion Two heterozygous NOBOX variants were identified in women with an OZEMA phenotype. Where pathogenic NOBOX variants are typically linked to POI, our clinical findings suggest that NOBOX plays a role in subsequent oocyte maturation and early embryo development.

STUDY QUESTION: Is there an association between different mitochondrial DNA (mtDNA) genotypes and female infertility or ovarian response, and is the appearance of variants in the oocytes favored by medically assisted reproduction (MAR) techniques? SUMMARY ANSWER: Ovarian response was negatively associated with global non-synonymous protein-coding homoplasmic variants but positively associated with haplogroup K; the number of oocytes retrieved in a cycle correlates with the number of heteroplasmic variants in the oocytes, principally with variants located in the hypervariable (HV) region and rRNA loci, as well as non-synonymous protein-coding variants. WHAT IS KNOWN ALREADY: Several genes have been shown to be positively associated with infertility, and there is growing concern that MAR may facilitate the transmission of these harmful variants to offspring, thereby passing on infertility. The potential role of mtDNA variants in these two perspectives remains poorly understood. STUDY DESIGN, SIZE, DURATION: This cohort study included 261 oocytes from 132 women (mean age: 32 ± 4 years) undergoing ovarian stimulation between 2019 and 2020 at an academic center. The oocyte mtDNA genotypes were examined for associations with the women’s fertility characteristics. PARTICIPANTS/MATERIALS, SETTING, METHODS: The mtDNA of the oocytes underwent deep sequencing, and the mtDNA genotypes were compared between infertile and fertile groups using Fisher’s exact test. The impact of the mtDNA genotype on anti-Müllerian hormone (AMH) levels and the number of (mature) oocytes retrieved was assessed using the Mann–Whitney U test for univariate analysis and logistic regression for multivariate analysis. Additionally, we examined the associations of oocyte maturation stage, infertility status, number of ovarian stimulation units, and number of oocytes retrieved with the type and load of heteroplasmic variants using univariate analysis and Poisson or linear regression analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Neither homoplasmic mtDNA variants nor haplogroups in the oocytes were associated with infertility status or with AMH levels. Conversely, when the relationship between the number of oocytes retrieved and different mtDNA genotypes was examined, a positive association was observed between the number of metaphase (MII) oocytes (P = 0.005) and haplogroup K. Furthermore, the presence of global non-synonymous homoplasmic variants in the protein-coding region was significantly associated with a reduced number of total oocytes and MII oocytes retrieved (P < 0.001 for both). Regarding the type and load of heteroplasmic variants in the different regions, there were no significant associations according to maturation stage of the oocyte or to fertility status; however, the number of oocytes retrieved correlated positively with the total number of heteroplasmic variants, and specifically with non-synonymous protein-coding, HV and rRNA variants (P < 0.001 for all). LIMITATIONS, REASONS FOR CAUTION: The current work is constrained by its retrospective design and single-center approach, potentially limiting the generalizability of our findings. The small sample size for specific types of infertility restricts this aspect of the findings. WIDER IMPLICATIONS OF THE FINDINGS: This work suggests that mitochondrial genetics may have an impact on ovarian response and corroborates previous findings indicating that the size of the oocyte cohort after stimulation correlates with the presence of potentially deleterious variants in the oocyte. Future epidemiological and functional studies based on the results of the current study will provide valuable insights to address gaps in knowledge to assess any prospective risks for MAR-conceived offspring. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Research Foundation Flanders (FWO, Grant numbers 1506617N and 1506717N to C.S.), by the Fonds Wetenschappelijk Fonds, Willy Gepts Research Foundation of Universitair Ziekenhuis Brussel (Grant numbers WFWG14-15, WFWG16-43, and WFWG19-19 to C.S.), and by the Methusalem Grant of the Vrije Universiteit Brussel (to K.S.). M.R. and E.C.d.D. were supported predoctoral fellowships by the FWO, Grant numbers 1133622N and 1S73521N, respectively. The authors declare no conflict of interests. TRIAL REGISTRATION NUMBER: N/A.

To prevent the transfer of embryos affected by monogenic conditions and/or chromosomal defects, preimplantation genetic testing (PGT) requires trophectoderm biopsy and cryopreservation. In 2–6% of biopsies, the diagnosis may be inconclusive due to DNA amplification failure or low-quality results. In these cases, a round of re-warming, re-biopsy, and re-cryopreservation is required to obtain a genetic diagnosis. In other cases, when the IVF centre starts providing PGT and/or when the patients develop an indication because of multiple failures, miscarriages or the birth of an affected child after IVF, cryopreserved untested embryos may be warmed, biopsied, and then re-vitrified. However, it is still unclear whether multiple manipulations may reduce reproductive outcomes after PGT. This study aimed at conducting a systematic review to investigate the available evidence on the safety of double biopsy and/or double cryopreservation–warming and provide recommendations in this regard. We performed meta-analyses of the differences in the reproductive outcomes (live birth per embryo transfer [LBR per ET], clinical pregnancy rate per ET [CPR per ET], and miscarriage rate per clinical pregnancy [MR per CP]) in double cryopreservation and single biopsy (CBC) or double biopsy and double cryopreservation (BCBC) flows vs the control single biopsy and single cryopreservation (BC) flow. Cryo-survival rates before ET and gestational and perinatal outcomes were also reported. PRISMA guidelines were followed to gather all available information from the literature (PubMed, Scopus, and Embase). We used Medical Subject Headings (MeSH) terms and a list of specific keywords relevant for the study question. We searched for original studies in humans, published in peer-reviewed journals in English up to April 2024. Four independent authors assessed the articles for inclusion. One included paper was retrieved from another source. A total of 4219 records were identified, and 10 studies were included in the meta-analysis. Certainty of evidence level ranged from low to moderate. Both the CBC and BCBC groups showed reduced reproductive outcomes compared to the control (BC). Specifically, live birth rates per embryo transfer were lower in the CBC group (OR: 0.56, 95% CI: 0.38–0.81, I² = 58%; six studies) and the BCBC group (OR: 0.51, 95% CI: 0.34–0.77, I² = 24%; six studies). CPR per ET were also lower in the CBC group (OR: 0.68, 95% CI: 0.51–0.92, I² = 57%; seven studies) and the BCBC group (OR: 0.60, 95% CI: 0.46–0.78, I² = 0%; seven studies). Additionally, MR per CPs were higher in both the CBC group (OR: 1.68, 95% CI: 1.02–2.77, I² = 50%; seven studies) and the BCBC group (OR: 2.08, 95% CI: 1.13–3.83, I² = 28%; seven studies). Cryo-survival as well as gestational and perinatal outcomes were within the expected norms in the studies reporting them. Improved genetic technologies, standardization of laboratory protocols, operators’ proficiency with biopsy and cryopreservation, and continuous monitoring of the performance are essential to minimize inconclusive diagnoses and the putative impact of additional embryo manipulations. Although poorer reproductive outcomes might result from double biopsy and/or double cryopreservations, these practices may still be worthwhile to avoid transferring affected/aneuploid blastocysts. Therefore, the risks must be weighed against the potential benefits for each specific couple. PROSPERO (ID: CRD42024503678) Double-cryopreservation single-biopsy (CBC) and double-biopsy double-cryopreservation (BCBC) may reduce the clinical pregnancy rate (CPR) and live birth rate (LBR) per embryo transfer (ET) and increase miscarriage rate (MR), compared with single-biopsy single-cryopreservation (BC). Image created with BioRender.

The field of reproductive genetics has undergone significant advancements with the completion of the Human Genome Project and the development of high-throughput sequencing techniques. This has led to the identification of numerous genes involved in both male and female infertility, revolutionizing the diagnosis and management of infertility patients. Genetic investigations, including karyotyping, specific genetic tests, and high-throughput sequencing, have become essential in determining the genetic causes of infertility. Moreover, the integration of genetics into reproductive medicine has expanded the scope of care to include not only affected individuals or couples but also their family members. Genetic consultations and counselling play a crucial role in identifying potentially affected relatives and offering tailored therapy and the possibility of fertility preservation. Despite the current limited therapeutic options, an increasing understanding of genotype–phenotype correlations in infertility genes holds promise for improved treatment outcomes. The availability of genetic diagnostic tools has reduced the number of idiopathic infertility cases by providing accurate aetiological diagnoses. The transition from research to clinical practice in reproductive genetics requires the establishment of genetic consultations and data warehousing systems to provide up-to-date information on gene–disease relationships. Overall, the integration of genetics into reproductive medicine has brought about a paradigm shift, emphasizing the familial dimension of infertility and offering new possibilities for personalized care and family planning.

Is there a risk factor association or a monogenic relationship between FMR1 premutation and developing Fragile X-associated primary ovarian insufficiency (FXPOI)? There is an association between FMR1 premutation and FXPOI rather than a monogenic relationship, which is highly dependent on ethnicity. Among the 40 genes involved in primary ovarian insufficiency (POI), identified in our recent systematic review (Van Der Kelen et al., 2022), the FMR1 premutation is considered as the most common cause of POI. A premutation in the FMR1 gene is defined as a CGG trinucleotide repeat length between 55 and 200 in the 5′ untranslated region. The term FXPOI is used for women with premutation in FMR1 who have a loss of normal function of the ovaries before the age of 40. There is conflicting evidence about the relationship between POI and length of premutation FMR1 alleles. Curated publications identified in PubMed and Web of Science on genetics of human female infertility and sex development by using the MESH terms, key words and inclusion/exclusion criteria described in our comprehensive systematic review were subjected to screening for “FMR1 gene.” The articles included cover a period from 1988 to the 1^st of November 2021. A total of 161 publications in PubMed and 61 unique publications in Web of Science were identified, and subsequently screened for triplet expansion, primary ovarian insufficiency, and/or early menopause. Of these, 56 papers were selected, excluding publications exclusively on males as well as studies on non-human species, pediatric cases, reviews, and expression studies. The study and control groups repeat numbers, ethnicity, and conclusions in the article are listed. Expansion in CGG trinucleotide repeat length happens at female meiosis and the risk for expansion increases with an increasing number of CGG repeats. There is an intermediate zone between 45 and 55 CGG repeats when the allele may be stable or unstable. Among 56 publications, 54 describing FMR1 triplet repeat size in women with POI, 34 were studying only the women with POI (observational) while 20 were including also control group (comparative). 2 publications were meta-analyses in which 13 and 11 case control studies were included respectively, 5 studies being in common. POI-associated premutations showed a wide range of repeat sizes. Women carrying midsize range repeats (>70-<100) potentially have a higher risk for POI compared to the general population. In recent decades, population-based screenings have indicated that FMR1 premutations are not as prevalent in women with ovarian insufficiency as previous estimates have suggested, but they still represent a substantial cause of POI. When present, the number of AGG interruptions and the size of uninterrupted CGG repeats are directly correlated with the ovarian reserve. No increased risk of POI associated with a premutation was reported among populations of non-European descent, such as the Han Chinese, Indian, and Jordan populations. Available reports are difficult to compare because the ethnicity and number of patients analyzed, availability of clinical data and the quality of results are different in each study. Additionally, an effect of X-inactivation in POI women with premutation was not studied. Owing to the low penetrance and a molecular mechanism that has not yet been fully elucidated, the clinical utility of FMR1 screening in women with reduced ovarian reserve needs further investigation before clinical implication. N/A

Study question: What is the predicted cumulative live birth rate (CLBR) relative to AMH-level for women undergoing preimplantation genetic testing (PGT) for monogenic disorders and structural rearrangements? Summary answer: Anti-müllerian hormone (AMH) and age allow to give an estimate of the CLBR in PGT and becomes poor when AMH is ≤ 2µg/L and age ≥42y. What is known already: Ovarian response is an important contributory factor in PGT in order to obtain a sufficient number of embryos for genetic analysis. A proportion of the obtained embryos will be carrier of the disease tested, and another small proportion of the embryos will fail to have a genetic diagnosis hence will not be available for embryo transfer. The number of embryos not suitable for embryo transfer as a result of PGT varies between 25-81% according to the indication for PGT. The combination of female age and AMH are good predictors for CLBR in conventional IVF. Study design, size, duration: This is a single-centre retrospective cohort study including 1,522 females undergoing 3,130 PGT cycles over a 6-year period (01/01/2015-31/12/2020) at a university-based referral centre for PGT. The principal outcome measure was CLBR per intention to treat (ITT), and the secondary outcome was live birth rate (LBR) per embryo transfer, in couples undergoing PGT-M and PGT-SR either by polymerase chain reaction (PCR), single nucleotide polymorphism (SNP) array, microarray-based comparative genomic hybridization (aCGH), or next-generation sequencing (NGS). Participants/materials, setting, methods: Mean ±SD was applied for continuous outcomes, whereas (relative) frequency was applied for dichotomous outcomes. Multivariable logistic regression analysis was applied with dependent variable being the CLBR or LBR and the independent variables the AMH, female age, BMI, inheritance mode and PGT-technique. Best-fit (Akaike information criterion (AIC)) versus polynomials were analysed. Non-inferiority testing between different technologies was performed. A 3D-prediction mosaic (including AMH, age and CLBR) was created, which can be used for counseling purposes. Main results and the role of chance: The mean female age is 32.46 years (SD 4.43), with a mean AMH level of 2.75 µg/L (SD 2.58) and a mean BMI of 24.24 (SD 4.43). Age and AMH significantly affect CLBR irrespective of the inheritance mode or PGT technique used. There is a gradual decline of CLBR from a female age of 25 years onwards reaching a critical threshold of less than 10% CLBR per ITT over the age of 42 with AMH levels ≤2 µg/L. Between cleavage stage biopsy techniques (PCR) and trophectoderm biopsy PGT techniques (SNP array, aCGH, NGS) used there was no significant difference in outcome per ITT, however per embryo transfer there is a significantly higher chance of live birth in SNP array, aCGH and NGS cycles compared to PCR cycles. Limitations, reasons for caution: Despite the large sample size, the findings are confined by limited confounder adjustment. Wider implications of the findings: In a PGT-program, couples need to be informed on the limited prognosis, if the combination of female age and AMH is unfavourable, in order to allow them to make other reproductive choices. The prediction mosaic produced in this study can be used as an insightful visual tool in counseling PGT-couples. Trial registration number: Not applicable

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. 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. 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. 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. 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. Current evidence supports using 105 genes in panels to screen for female infertility and differences in sex development.

(Abstracted from Hum Reprod 2020;35(11):2488–2496) Despite advances in preimplantation genetic testing (PGT), inconclusive diagnoses following aneuploidy screening, chromosomal rearrangement, or monogenic testing do occur regularly. With PGT these days shifting toward more and more trophectoderm (TE) biopsy, test failures inevitably involve a second round of biopsy and blastocyst vitrification.

INTRODUCTION AND AIMS: Polycystic kidney disease (PKD) comprises a group of monogenic disorders that result in renal cyst development. In this study, the strategies and clinical outcome of a large cohort of preimplantation genetic testing (PGT) cycles for PKD performed at the Centre for Medical Genetics of the Universitair Ziekenhuis Brussel between 2005 and 2016 are reported. As males affected with autosomal dominant polycystic kidney disease (ADPKD) may present with reproductive system abnormalities and infertility, the clinical outcome was compared between couples with the female partner affected with ADPKD and couples with the male partner affected with ADPKD.