Human Reproduction

ObjectiveTo investigate the possible impact of Pfizer-BioNTechs mRNA BNT162b2 COVID-19 vaccine on womens fertility. MethodsA retrospective single-center study examining womens IVF treatment parameters and pregnancies before and after their vaccination between February and May 2021. Each woman served as a self-control before and after vaccination. Additionally, in order to neutralize the effect of the sperm on fertilization, only Intracytoplasmic Sperm Injection (ICSI) patients who were currently being treated with an ICSI cycle and had an earlier ICSI cycle available were included in the study. The study outcomes compared between the PRE and POST vaccination groups and consisted of: the IVF cycle outcomes, including the number of oocytes retrieved; the number of matured oocytes; the fertilization rate; and the number and quality of embryos at day 3. Clinical pregnancy was based on the first hCG value reported if the data were available for both cycles. ResultsA final total of 47 women were eligible for inclusion with a mean interval of 362 {+/-}368 days between the two ovum pick ups. The characteristics of their ICSI cycles before and after the vaccination were similar for all the parameters. Additionally, the number and percentage of clinical pregnancies did not significantly differ between the PRE and POST vaccination groups (n=15). ConclusionThis study is the first to evaluate the impact of the BNT162b2 vaccine on womens fertility. From our findings, the vaccine appears to have no impact on womens fertility. This study is the first step in abolishing the misinformation derived from unreliable sources and reassuring patients in order to improve compliance and promote COVID-19 eradication.

BackgroundPolygenic embryo screening (PES) has recently become available to in-vitro fertilization (IVF) patients, allowing them to evaluate the genetic risk of each of their embryos for polygenic conditions such as heart attack or diabetes. Initial modeling predicted that transferring the embryo with the lowest genetic risk for one or more diseases would substantially reduce prevalence in the next generation, with relative risk reductions up to 50%. However, these models assumed the availability of a prespecified number of embryos and that the embryo with the most favorable polygenic risk is born once transferred to the uterus. In reality, a large percentage of embryo transfers do not lead to live births, and IVF frequently results in no or only a single live birth. MethodsTo quantify the expected risk reduction in the context of IVF, we used two datasets: 6944 ovarian stimulation cycles from 4452 Italian infertility patients and 2138 stimulation cycles of egg donors. In both datasets, we simulated the hypothetical application of PES in these cycles by assigning patients and their embryos randomly drawn polygenic risk scores for a given disease, assuming that embryos have been transferred in increasing order of their risk, and tracing their birth outcomes. We then compared the risk of the embryo born after hypothetical PES to the risk of an embryo born without PES. We either considered only completed cycles or integrated over possible birth outcomes of non-transferred embryos in incomplete cycles. ResultsIn stimulation cycles in infertility patients in which all embryos have been transferred and at least one child was born, we estimate that PES will result in relative risk reductions of just {approx}1-3%. In an intention-to-screen analysis of all completed cycles (regardless of birth outcomes), relative risk reductions are under 0.5%. The risk reductions increase, as expected, with more euploid blastocysts and with younger maternal age. Including incomplete cycles (in which not all embryos have been transferred) increases risk reductions to {approx}2-5%, due to the availability of more euploid blastocysts and a higher live birth rate per transfer in these cycles. Pooling all embryos from all cycles of the same patient increases risk reductions to {approx}5-10%. Relative risk reductions in egg donor cycles reach {approx}20% even with a single stimulation cycle per donor. ConclusionsWith the exception of particularly good-prognosis patients or cycles, typical infertility patients would benefit little from PES. In fertile patients, as represented by egg donors, PES is predicted to achieve greater relative risk reductions. However, even though these reductions are still substantially lower than prior estimates that did not account for realistic live birth rates. Ethical, social, and clinical issues associated with offering PES in the general population should be prioritized in future research.

Study questionHow are Medically Assisted Reproduction (MAR) treatments (Fertility enhancing drugs (FED), artificial/intrauterine insemination (AI/IUI)), assisted reproductive technology (ART) with autologous/donor oocytes) associated with maternal morbidity (MM)? Summary answerMore invasive MAR treatments (ART and AI/IUI) are associated with higher risk of MM, whilst less invasive treatments are not; this relationship is partially explained by higher prevalence of multifetal gestation and obstetric comorbidities in women undergoing more invasive treatment, but the persistent association suggests subfertility itself may contribute to maternal morbidity risk. What is known alreadyWomen conceiving through MAR are at higher risk of MM, however, reported risks vary depending on the measurement of MM and data available on confounding. Study design, size, durationBirth certificates were used to study maternal morbidity among all women giving birth in Utah, U.S., between 2009 and 2017 (N=460,976 deliveries); 19,448 conceived through MAR (4.2%). The MM outcome measure included the presence of any of the following: blood transfusion; unplanned operating room procedure; admission to ICU; eclampsia; unplanned hysterectomy; ruptured uterus. Participants/materials, setting, methodsLogistic regressions were estimated for the binary outcome (presence of any of the MM conditions). We assessed MM among women conceiving through MAR (overall and by type of treatment) compared to those conceiving spontaneously in the overall sample before and after adjustment for maternal socio-demographic characteristics (maternal age, family structure, level of education, Hispanic origin, parity), pre-existing maternal comorbidities (i.e., chronic hypertension, heart disease, asthma), multifetal gestation, and obstetric comorbidities (i.e., placenta previa, placental abruption, preterm delivery, cesarean delivery). Main results and the role of chanceWomen conceiving through MAR had higher risk of MM; however, the magnitude of the association differed depending on the type of treatment. In the unadjusted models, more invasive treatments were associated with higher odds of MM: OR 5.71 (95% CI 3.50-9.31) among women conceiving through ART with donor oocytes, OR 3.20 (95% CI 2.69-3.81) among women conceiving through ART with autologous oocytes, and OR 1.85 (95% CI 1.39-2.46) among women conceiving through AI/IUI, whereas women conceiving through FED had similar risks of MM to compared to women conceiving spontaneously (SC), OR 1.09 (95% CI 0.91-1.30). The associations between MAR and MM were largely attenuated once multifetal gestation was accounted for. After controlling for obstetric comorbidities, the associations were further attenuated, yet the coefficients remained higher among women conceiving through ART with either donor oocytes OR 1.70 (95% CI 0.95-3.04) or autologous oocytes OR 1.46 (95% CI 1.20-1.78) compared to women conceiving spontaneously. In analyses limited to singleton pregnancies, the differences in MM between women conceiving through MAR and SC were smaller in the unadjusted models. Nevertheless, women conceiving through more invasive treatments exhibited higher risk of MM. After adjusting for obstetric comorbidities, the coefficients were further attenuated and statistically insignificant for all types of treatments. Limitations, reasons for cautionThe data do not allow us to separate the confounding effects of subfertility on maternal morbidity from those of MAR treatments per se as there is no information on the history of previous infertility treatments or length of trying to become pregnant prior to conception. Our data also do not permit us to distinguish among different ART treatment approaches that could change certain risks (e.g. fresh or frozen embryo transfer, intracytoplasmic sperm injection, or preimplantation genetic screening via blastocyst sampling). Wider implications of the findingsOur findings showing that more invasive MAR treatments are associated with higher MM suggest that subfertility could be an important unobserved factor in MM risk as it could be associated with both higher risk of MM and with undergoing more invasive procedures. Though the odds of MM were generally lower or non-significant after accounting for multifetal gestation, there remain important clinical implications because a high proportion of individuals undergoing MAR in Utah have multiple births. Therefore, the association between MAR, multifetal gestation, and MM may play a role in counselling and patient and clinician choice of MAR therapies. Study funding/competing interest(s)This work was supported by European Research Council agreement n. 803958 (to A.G.). Authors have no conflict of interest to declare. MM was supported by the Strategic Research Council (SRC), FLUX consortium, decision numbers 345130 and 345131; by the National Institute on Aging (R01AG075208); by grants to the Max Planck - University of Helsinki Center from the Max Planck Society (Decision number 5714240218), Jane and Aatos Erkko Foundation, Faculty of Social Sciences at the University of Helsinki, and Cities of Helsinki, Vantaa and Espoo; and the European Union (ERC Synergy, BIOSFER, 101071773). Views and opinions expressed are, however, those of the author only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them. We thank the Pedigree and Population Resource of Huntsman Cancer Institute, University of Utah (funded in part by the Huntsman Cancer Foundation) for its role in the ongoing collection, maintenance and support of the Utah Population Database (UPDB). We also acknowledge partial support for the UPDB through grant P30 CA2014 from the National Cancer Institute, University of Utah and from the University of Utahs program in Personalized Health and Utah Clinical and Translational Science Institute. MPD receives salary support from the March of Dimes and the American Board of Obstetrics and Gynecology as part of the Reproductive Scientist Development Program, as well as NICHD 1U54HD113169 and NIMHD 1R21MD019175-01A1. Trial registration numbernot applicable

BackgroundNext generation sequencing (NGS) has increased detection sensitivity of intermediate chromosome copy number variations (CNV) consistent with chromosomal mosaicism. Recently, this methodology has found application in preimplantation genetic testing (PGT) of trophectoderm (TE) biopsies collected from IVF-generated human embryos. As a consequence, the detection rate of intermediate CNV states in IVF embryos has drastically increased, posing questions about the accuracy in identifying genuine mosaicism in highly heterogeneous biological specimens. The association between analytical values consistent with mosaicism and the reproductive potential of the embryo, as well as newborns chromosomal normalcy, have not yet been thoroughly determined. MethodsWe conducted a multicentre, double-blinded, non-selection trial including 1,190 patients undergoing in a total of 1,337 IVF with preimplantation genetic testing for aneuploidies (PGT-A) treatment cycles. NGS was performed on clinical TE biopsies collected from blastocyst-stage embryos. All embryos were reported as euploid if all autosomes had a chromosomal copy number value below the threshold of 50% abnormal cells per sample. After embryo transfer, three comparative classes were analysed: uniformly euploid profiles (<20% aneuploid cells), putative low-degree mosaicism (20%-30% aneuploid cells) or putative moderate-degree mosaicism (30%-50% aneuploid cells). Primary outcome measure was live birth rate (LBR) per transfer and newborns karyotype. ResultsLBR after transfer of uniformly euploid embryos, low-degree, and moderate-degree mosaic embryos were 43.4% (95% C.I. 38.9 - 47.9), 42.9% (95% C.I. 37.1 - 48.9) and 42% (95% C.I. 33.4 - 50.9), respectively. No difference was detected for this primary outcome between euploid and mosaic low/moderate categories (OR= 0.96; 95% CI 0.743 to 1.263; P=0.816). The non-inferiority endpoint was met as the confidence interval for the difference fell below the planned 7.5% margin (95% C.I. -5.7 - 7.3). Likewise, no statistically significant difference was observed comparing moderate versus low degree mosaic embryos (P=0.92). Neonatal karyotypes were also similar and no instances of mosaicism or uniparental disomies (UPDs) were detected in babies born following putative low or moderate-degree mosaic embryo transfer. Should the embryos with low or moderate-degree mosaic TE biopsies had been classified as chromosomally abnormal and thus discarded for clinical use, LBR per cycle would have decreased by 36% without any clinical benefit. ConclusionsThis prospective non-selection trial provides substantial evidence that reporting and/or not transferring embryos with low/moderate-degree mosaicism for whole chromosomes have no clinical utility. Moreover, dismissing these embryos from clinical use has the counterproductive effect of reducing overall embryo availability, thus reducing the chance of successful outcome derived from an IVF treatment without any clinical benefit. (Funded by Igenomix; ClinicalTrials.gov number, NCT03673592)

ObjectiveTo evaluate how minimal controlled ovarian stimulation (COS) for in vitro maturation (IVM) affects subjects oocyte retrieval experiences compared to conventional COS, considering side effects DesignRetrospective Survey Study SettingClinical in vitro fertilization (IVF) treatment centers in Spain and the United States. SubjectsData were collected from subjects undergoing minimal COS (n=110; 600-800 IU FSH) for IVM and conventional COS for egg donation (n=48; 2000-3000 IU FSH) from April 2022 to November 2023. In the same period, a pairwise comparison of subjects (n=13) undergoing both minimal COS for IVM and conventional COS for oocyte cryopreservation was conducted. Intervention/ExposureMinimal and conventional controlled ovarian stimulation. Main Outcome MeasuresThe most common side effects suffered during ovarian stimulation and after OPU, satisfaction level, and the likelihood of recommending or repeating minimal or conventional COS. Statistical analysis included Mann Whitney, Wilcoxon, Chi-square, and McNemar tests, with a significance level set at p<0.05. ResultsDuring minimal COS, most subjects did not experience breast swelling (86%), pelvic or abdominal pain (76%), nausea or vomiting (96%), and bleeding (96%). After oocyte pick-up, the majority (75%) reported no pelvic or abdominal pain. The most common side effect was abdominal swelling (52%). Compared to conventional COS cycles, minimal COS subjects reported significantly less post-retrieval pain, with 33% experiencing no pain (vs. 6%; p=0.0011) and with a reduced severe level of pain (5% vs.19%; p=0.0097), leading to fewer subjects requiring pain medication (25% vs. 54%; p=0.0003). Additionally, 85% of women were very satisfied with minimal stimulation and would recommend or repeat the treatment. In the comparison in which each donor underwent both minimal and conventional COS treatments, women indicated more side effects with the conventional stimulation, presenting a significantly overall higher level of pain (p=0.0078). ConclusionReducing the hormonal dose for ovarian stimulation has a beneficial effect on subjects, suggesting the combination of minimal COS with IVM techniques is a well-tolerated alternative for women who cannot or do not wish to undergo conventional controlled ovarian hyperstimulation.

STUDY QUESTIONWhich mechanisms of action and candidate drugs can be used to treat endometrial failure caused by molecular alterations rather than endometrial timing? SUMMARY ANSWERGenistein, pioglitazone, alprostadil, flunisolide, and tenoxicam emerged as potential therapies to treat two molecular causes of endometrial failure not originating in endometrial timing. WHAT IS KNOWN ALREADYSeveral studies have described molecular profiles of endometrial failure unrelated to endometrial timing and proposed diagnostic tools based on clinical and transcriptomic data, but effective therapeutic options remain lacking. Hence, there is a pressing need for tailored treatments to enable personalised medicine in endometrial-factor infertility. STUDY DESIGN, SIZE, DURATIONThis multicentre prospective study, conducted at 5 fertility clinics in Spain between January 2019 and August 2022, included 192 patients undergoing in vitro fertilisation with hormone replacement therapy, whose endometrial biopsies were collected during the mid-secretory phase. PARTICIPANTS/MATERIALS, SETTING, METHODSOf 291 endometrial biopsies, 192 met the quality criteria and 161 were classified according to clinical and transcriptomic data using a semi-supervised learning model for prognosis. Before classification, transcriptomic variation related to endometrial timing was corrected using our validated transcriptomic endometrial-dating model. Profiles were analysed using systems pharmacology approaches combining network analysis and reversal signature matching to identify therapeutic drugs capable of reversing molecular disruption. Candidate drugs were grouped by mechanism of action and prioritised by side-effect profile. Selected drugs were validated in endometrial cells through RT-PCR, F-actin staining, and enzyme-linked immunosorbent assays. MAIN RESULTS AND THE ROLE OF CHANCEFour transcriptomic profiles were identified using artificial intelligence models, each with distinct clinical implications. Two profiles were associated with poor prognosis: clinical miscarriage-associated (CMA, n = 27) and biochemical miscarriage-associated (BMA, n = 16). CMA was characterised by upregulation of differentiation-related genes and BMA by upregulation of immune-related genes. The 4 profiles were homogeneous in demographic and embryological parameters (age, BMI, and embryo quality), reinforcing their biological relevance. Approved drugs capable of reversing these disrupted expression patterns were identified. Both BMA and CMA were linked to abnormal decidualisation, while BMA also showed immune dysregulation. Genistein and pioglitazone promoted decidualisation in vitro, whereas alprostadil, flunisolide, and tenoxicam inhibited immune responses in endometrial cell cultures, supporting their potential therapeutic role in endometrial failure not originating in endometrial timing. LIMITATIONS, REASONS FOR CAUTIONAlthough our artificial intelligence-based stratification model revealed clinical and functional differences among profiles, it was designed for drug repurposing rather than predictive diagnosis. A larger, specifically designed study would be required to validate predictive performance and generalisability. Further clinical trials are needed to evaluate the proposed drugs as personalised treatments for this condition. WIDER IMPLICATIONS OF THE FINDINGSThis is the first application of a systems-based drug repurposing strategy in IVF to develop tailored therapeutic interventions. We propose genistein, pioglitazone, alprostadil, flunisolide, and tenoxicam as approved, safe drugs identified through an evidence-based approach that could prevent loss of good-quality embryos and miscarriage due to maternal endometrial factors. These findings, supported by functional in vitro validation, pave the way for future clinical trials advancing personalised medicine in endometrial-factor infertility. TRIAL REGISTRATION NUMBERNot applicable

Proper action of the female sex steroids, 17{beta}-estradiol (E2) and progesterone (P4) on endometrium is essential for fertility. Beyond its role in regulating the cell cycle, cyclin A2 (CCNA2) also mediates E2 and P4 signaling in vitro, but a potential role in modulating steroid action for proper endometrial tissue development and function is unknown. To fill this gap in our knowledge, we examined human endometrial tissue from fertile and infertile women for CCNA2 expression and correlated this with pregnancy outcome. Functional assessment of CCNA2 was validated in vivo using a conditional Ccna2 uterine deficient mouse model while in vitro function was assessed using human cell culture models. We found that CCNA2 expression was significantly reduced in endometrial tissue, specifically the stromal cells, from women undergoing in vitro fertilization who failed to achieve pregnancy. Conditional deletion of Ccna2 from moue uterine tissue recapitulated the inability to achieve successful pregnancy which appears to be due to alterations in the process of decidualization, which was confirmed using in vitro models. From these studies, we conclude that CCNA2 expression during the proliferative/regenerative stage of the menstrual cycle acts as a safeguard allowing for proper steroid responsiveness, decidualization and pregnancy. When CCNA2 expression levels are insufficient there is impaired endometrial responsiveness, aberrant decidualization and loss of pregnancy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/545284v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@150b471org.highwire.dtl.DTLVardef@176a804org.highwire.dtl.DTLVardef@110a47dorg.highwire.dtl.DTLVardef@19f186c_HPS_FORMAT_FIGEXP M_FIG C_FIG

STUDY QUESTIONCan a large-scale genome-wide association study (GWAS) meta-analysis identify the genomic risk loci and associated candidate genes for female genital tract (FGT) polyps, provide insights into the mechanism underlying their development, and inform potential overlap with other traits, including endometrial cancer? SUMMARY ANSWERGWAS meta-analysis of FGT polyps highlighted the potentially shared mechanisms between polyp development and cancerous processes. WHAT IS KNOWN ALREADYSmall-scale candidate gene studies have focused on biological processes such as estrogen stimulation and inflammation to clarify the biology behind FGT polyps. However, the exact mechanism for the development of polyps is still elusive. At the same time, a genome-wide approach, which has become the gold standard in complex disease genetics, has never been used to uncover the genetics of the FGT polyps. STUDY DESIGN, SIZE, DURATIONWe performed a genome wide association study (GWAS) meta-analysis including a total of 25,100 women with FGT polyps (International Classification of Disease, ICD-10 diagnosis code N84) and 207,193 female controls (without N84 code) of European ancestry from the FinnGen study (11,092 cases and 94,394 controls) and the Estonian Biobank (EstBB, 14,008 cases and 112,799 controls). PARTICIPANTS/MATERIALS, SETTING, METHODSA meta-analysis and functional annotation of GWAS signals were performed to identify and prioritise genes in associated loci. To determine associations with other phenotypes, we performed a look-up of associated variants across multiple traits and health conditions, a genetic correlation analysis, and a phenome-wide association study (PheWAS) with ICD10 diagnosis codes. MAIN RESULTS AND THE ROLE OF CHANCEOur GWAS meta-analysis revealed ten significant (P < 5 x 10-8) genomic risk loci. Two signals, rs2277339 (P = 7.6 x 10-10) and rs1265005 (P = 1.1 x 10-9) (in linkage disequilibrium (LD) with rs805698 r2 = 0.75), are exonic missense variants in PRIM1, and COL17A1 genes, respectively. Based on the literature, these genes may play a role in cellular proliferation. Several of the identified genomic loci had previously been linked to endometrial cancer and/or uterine fibroids. Thus, highlighting the potentially shared mechanisms underlying tissue overgrowth and cancerous processes, which may be relevant to the development of polyps. Genetic correlation analysis revealed a negative correlation between sex hormone-binding globulin (SHBG) and the risk of FGT polyps (rg = -0,21, se = 0.04, P = 2.9 x 10-6), and on the phenotypic level (PheWAS), the strongest associations were observed with endometriosis, leiomyoma of the uterus and excessive, frequent and irregular menstruation. LARGE SCALE DATAThe complete GWAS summary statistics will be made available after publication through the GWAS Catalogue (https://www.ebi.ac.uk/gwas/). LIMITATIONS, REASONS FOR CAUTIONIn this study, we focused broadly on polyps of FGT and did not differentiate between the polyp subtypes. The prevalence of FGT polyps led us to assume that most women included in the study had endometrial polyps. Further study on the expression profile of FGT polyps could complement the GWAS study to substantiate the functional importance of the identified variants. WIDER IMPLICATIONS OF THE FINDINGSThe study findings have the potential to significantly enhance our understanding of the genetic mechanisms involved, paving the way for future functional follow-up, which in turn could improve the diagnosis, risk assessment, and targeted treatment options, since surgery is the only line of treatment available for diagnosed polyps. TRIAL REGISTRATION NUMBERNot applicable

STUDY QUESTION[Do structural genomic variants, that can be identified by using optical genome mapping, contribute to male infertility?] SUMMARY ANSWER[By using optical genome mapping we can identify several types of structural variants, both known and new, that may contribute to male infertility.] WHAT IS KNOWN ALREADY[Traditional approaches such as karyotyping, CFTR and chromosome Y microdeletion testing are successful in explaining clinical findings in [~]30% of MI patients, leaving the rest without a genetic diagnosis. Recent research suggests at least 265 genes may play a role in male fertility. While the assessment of the roles of copy number variants and single nucleotide variants in monogenic forms of disease in these genes is underway, much less is known about structural variants.] STUDY DESIGN, SIZE, DURATION[We performed a longitudinal case/control study on a total of 220 individuals; 88 patients with male infertility, negative for cytogenetic abnormalities using karyotyping, and molecular testing for chrY microdeletions, and CFTR gene variants, and 132 healthy male individuals that underwent optical genomic mapping for other reasons. Exclusion criteria for the control cohort were low-sperm quality and/or inclusion in IVF procedures. The study was approved by the National Medical Ethics Committee of the Republic of Slovenia (reference number: 0120-213/2022/6). Optical genome mapping was performed from an aliquot of whole blood collected for routine testing purposes at the Clinical Institute of Genomic Medicine (CIGM), UMC Ljubljana from January 2023 to November 2024.] PARTICIPANTS/MATERIALS, SETTING, METHODS[We examined structural variants in 220 participants by using optical genome mapping, which was performed with DLE-1 SP-G2 chemistry and the Saphyr instrument. The de novo assembly and Variant Annotation Pipeline were executed on Bionano Solve3.7_20221013_25 while reporting and direct visualization of structural variants was done on Bionano Access 1.7.2. All obtained variants were filtered using the Bionano Access software and in-house generated gene/regions of interest panel bed files. The first filter was applied to include variants below a population frequency of 10%, and overlapping the regions of interest. Subsequently, all variants occurring with frequency 0% in the internal manufacturer variant dataset were manually evaluated for possible involvement of the overlapping genes or regions in biological processes involved in MI. The male infertility cohort also underwent research whole exome analyses as previously reported. All results of optical genomic mapping were confirmed by an appropriate alternative method where available.] MAIN RESULTS AND THE ROLE OF CHANCE[We show that the overall number of structural variants in MI patients does not differ from that of healthy individuals. By looking in detail at genes and regions associated with MI, we identified 21 rare variants absent from controls in 25.0 % of MI patients, of which five were likely causative, and two would be missed by using traditional approaches. These variants include inversions, duplications, amplifications, deletions (e.g. SPAG1), and insertions/expansions (e.g. DMPK), that were validated using additional methods. While the remaining SV cannot be currently classified as pathogenic according to existing criteria, they open a new avenue in genetic research of MI. LARGE SCALE DATA[Variants reported in this study were deposited into ClinVar under accession numbers SUB15650956 (https://www.ncbi.nlm.nih.gov/clinvar/)] LIMITATIONS, REASONS FOR CAUTION[Technical limitations of optical genome mapping include the lack of DLE-1 labelling of centromeric and telomeric regions, the inability to detect Robertsonian translocations, the unclear exact location of smaller structural variants located between the DLE-1 labels, and unclear boundaries in case of their location in segmentally duplicated regions (this limitation is shared with other methods). The ACGM criteria of rarity are also hard to apply, as the fertility status of the individuals in healthy population databases such as GnomAD and DGV is unknown. Similarly, gene-associated phenotype and the proposed inheritance model both need to be considered as parts of the ACMG criteria, but for many candidate genes associated with MI, no model of inheritance has yet been proposed.] WIDER IMPLICATIONS OF THE FINDINGS[Currently, with the established diagnostic approaches we are able to resolve [~]30% of male infertility cases, with [~]70% of patients remaining undiagnosed. The significance of our work is in showing that rare structural variants can be identified in MI, by using optical genome mapping, opening new avenues of research of the genetics of this important contributor to human fertility.] STUDY FUNDING/COMPETING INTEREST(S)[All authors declare having no conflict of interest in regard to this research. This work was funded by the Slovenian Research and Innovation Agency (ARIS) Programme grant P3-0326: Gynecology and Reproduction: Genomics for personalized medicine] Lay summaryMale infertility affects about 5% of adult males and has complex causes, including genetic ones, such as mutations in the CFTR gene, small deletions on chromosome Y, and balanced translocations, but currently we can only find a genetic cause in [~]30% of patients. This means [~]70% of cases remain undiagnosed but potentially, they too may have a yet unknown genetic cause. Indeed, so far research has shown at least 265 genes have been proposed to play a role in male fertility. In these genes, there has so far been limited research of single nucleotide variants and of copy number variants, but many structural variants are not visible using commonly used methods in clinical genetic testing. Therefore, apart from chromosome Y microdeletions and chromosomal numerical and structural anomalies, such as balanced translocations, the role of smaller structural variants in male infertility is unknown, but based from what we know from other diseases, they also may play a role in male infertility. Optical genome mapping is a novel method for the detection of structural variants, such as balanced and unbalanced translocations, insertions, duplications, deletions, and complex structural rearrangements in a wide range of sizes. By using optical genome mapping to test a cohort of 88 infertile men and 132 healthy controls, we aimed to provide the first insights into the range of SV that may be associated with MI. We found, by using optical genome mapping, the overall number of structural variants in MI patients not to be significantly different to the control group. However, by looking at genes and regions associated with MI, we can find rare structural variants that are absent from controls in 25.0% of MI patients. These variants include inversions, duplications, amplifications, deletions (e.g. deletion in SPAG1), and insertions/expansions (e.g. in DMPK), that were validated using additional methods. Five of these variants (5.6%) were likely causative, and two would be missed by traditional approaches. While the remaining SV cannot be currently classified as pathogenic according to existing criteria, they open a new avenue in genetic research of MI.

Study questionDoes the oestrogen receptor isoform, ER46, contribute to regulation of endometrial function?\n\nSummary answerER46 is expressed in endometrial tissues during the proliferative and secretory phases and is the predominant ER isoform in first trimester decidua. ER46 is abundantly expressed in uterine NK (uNK) cells and localised to the cell membrane. Activation of ER46 regulates the function of human uNK cells by increasing cell motility.\n\nWhat is known alreadyOestrogens acting via their cognate receptors are essential regulators of endometrial function and play key roles in establishment of pregnancy. ER46 is a 46kDa truncated isoform of full length ER (ER66, encoded by ESR1) that contains both ligand and DNA binding domains. Expression of ER46 in human endometrium has not been investigated previously. ER46 is located at the cell membrane of peripheral blood leukocytes and mediates rapid responses to oestrogens. UNK cells are a phenotypically distinct (CD56brightCD16-) population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. We have shown that oestrogens stimulate rapid increases in uNK cell motility. Previous characterisation of uNK cells suggests they are ER66-negative but expression of ER46 has not been characterised. We hypothesise that uNK cells express ER46 and that rapid responses to oestrogens are mediated via this receptor.\n\nStudy design, size, durationThis laboratory-based study used primary human endometrial (n=24) and decidual tissue biopsies (n=30) as well as uNK cells which were freshly isolated from first trimester human decidua (n=18).\n\nParticipants/materials, setting, methodsPrimary human endometrial and first trimester decidual tissue biopsies were collected using methods approved by the local institutional ethics committee (LREC/05/51104/12 and LREC/10/51402/59). The expression of oestrogen receptors (ER66, ER46 and ER{beta}) was assessed by qPCR, western blot and immunohistochemistry. Uterine Natural Killer (uNK) cells were isolated from first trimester human decidua by magnetic bead sorting. Cell motility of uNK cells was measured by live cell imaging: cells were treated with oestradiol (E2)-BSA (10nM equivalent), the ER{beta}-selective agonist 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; 10nM) or vehicle control (DMSO).\n\nMain results and the role of chanceER46 was detected in proliferative and secretory phase tissues and was the predominant ER isoform in first trimester decidua samples. Immunohistochemistry revealed ER46 was co-localised with ER66 in cell nuclei during the proliferative phase but detected in both the cytoplasm and cell membrane of stromal cells in the secretory phase and in decidua. Triple immunofluorescence staining of decidua tissues identified expression of ER46 in the cell membrane of CD56-positive uNK cells which were otherwise ER66-negative. Profiling of isolated uNK cells confirmed expression ER46 and localised ER46 protein to the cell membrane. Functional analysis of isolated uNK cells using live cell imaging demonstrated that activation of ER46 with E2-BSA significantly increased uNK cell motility.\n\nLimitations, reasons for cautionExpression patterns in endometrial tissue was only determined using samples from proliferative and secretory phases. Assessment of first trimester decidua samples was from a range of gestational ages which may have precluded insights into gestation specific changes in these tissues. Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in situ.\n\nWider implications of the findingsE2 is an essential regulator of reproductive competence. This study provides the first evidence for expression of ER46 in human endometrium and decidua of early pregnancy. We describe a mechanism for regulating the function of human uNK cells via expression of ER46 and demonstrate that selective targeting with E2-BSA regulates uNK cell motility. These novel findings identify a role for ER46 in human endometrium and provide unique insight into the importance of membrane-initiated signalling in modulating the impact of E2 on uNK cell function in women.\n\nStudy funding/competing interest(s)These studies were supported by MRC Programme Grants G1100356/1 and MR/N024524/1 to PTKS. HODC was supported by MRC grant G1002033.

ObjectiveTo study the effect of a one-step warming technique on survival and clinical outcomes of vitrified-warmed blastocysts on a consecutive cohort of patients. DesignRetrospective consecutive cohort study. SubjectsThis study included 1402 transferred embryos from 989 unique patients that were treated in 3 clinics. ExposureThe exposure group included embryos warmed using a one-step warming protocol of 1M sucrose solution for 1 minute. The control group included embryos warmed using traditional, multi-step warming, where embryos were exposed to 1M sucrose for 1 min, followed by 3 min in 0.5M sucrose, and 10 mins in washing solutions. Main Outcome MeasuresThe goal of this study was to compare survival, clinical pregnancy (CPR) and ongoing pregnancy (OPR) rates between multi- and one-step warming techniques. Additionally, subgroup analyses by maternal age, embryo morphology, day of vitrification and mode of fertilization were also performed. ResultsSurvival rates were comparable across all comparisons. Pregnancy rates were comparable between multi-step and one-step groups (CPR: 42.6% vs 44.3%, p=0.78; OPR: 33.2 %vs 37.5%, p=0.21). Pregnancy probabilities between warming techniques were comparable between groups at any age point (32 - CPR: 43.0% vs 47.7%; OPR: 34.5% vs 39.5; p>0.05; 42 - CPR: 24.2% vs 19.3%; OPR: 13.5% vs 13.5%; p>0.05). Good quality embryos (G2) had a lower chance of pregnancy than top quality embryos (G1) overall, but pregnancy rates were similar between groups (G1 - CPR: 52.3% vs 54.6%; OPR: 46.0% vs 48.1%; p>0.05; G2 - CPR: 38.6% vs 40.0%; OPR: 27.8% vs 33%; p>0.05). Similarly, Day 6 embryos were less likely to achieve pregnancy than Day 5 embryos, but pregnancy rates were comparable between groups (D5 - CPR: 44.8% vs 46.5%; OPR: 35.3% vs 40.0%; p>0.05; D6 - CPR: 28.0% vs 31.2%; OPR: 18.3% vs 23.4%; p>0.05). Pregnancy rates between ICSI for male factor infertility and IVF were again comparable between groups (ICSI - CPR: 40.9% vs 38.3%; OPR: 33.7% vs 32.5%; IVF - CPR: 45.0% vs 48.0%; OPR: 37.3% vs 41.9%; p>0.05). ConclusionOne-step embryo warming provides similar survival and pregnancy outcomes compared to classical multi-step warming while decreasing the procedure time by more than 90%.

Study questionDoes glucose concentration in culture medium have an impact on the DNA methylome of the early human embryo? Summary answerGlucose concentration is associated with changes in gene expression, global DNA methylation, methylation levels at CpG islands and at key histone modifications in human blastocysts. What is known alreadyPreimplantation human embryos are highly sensitive to their local environment, and this may have long term implications for the health of the developing embryo, fetus and offspring. Glucose is a standard component of human embryo culture media, due to its importance as a nutrient. However, concentrations of glucose differ widely between different commercially available types. The present study was designed to determine whether changes in glucose concentration could influence global methylation and gene expression in the human preimplantation embryo. Study design, size, durationHuman embryos were cultured in clinically relevant concentrations of glucose and global DNA methylation analysis was performed. The effect of glucose concentration on the embryo epigenome, specifically DNA methylation, was analysed. Participants/materials, setting, methodsHuman embryos surplus to treatment requirements were donated with informed consent from several ART centres. Embryos were cultured to the blastocyst stage in Vitrolife G-TL medium, either at 0.9 mM or 3.5 mM glucose, separated via immunosurgery into Inner Cell Mass (ICM) and trophectoderm (TE) samples, and compared for both DNA methylation and gene expression. This allowed us to evaluate the association between DNA methylation and previously importantly identified biological pathways. Main results and the role of chanceThe concentration of glucose in human embryo culture medium was associated with changes in gene expression and global DNA methylation in both ICM and TE, and methylation levels at CpG islands and key histone modifications. These results are significant because glucose is a major nutrient metabolised by human embryos in culture, and yet we know relatively little of its downstream effects on the genome and epigenome. Wider implications of the findingsCommercially available embryo culture media with varying glucose levels have also been associated with altered fetal growth, birthweight and postnatal development of IVF offspring. Our findings may have important ramifications for potential clinical markers of embryo quality and pregnancy initiation, and improve understanding of the mechanisms underlying the impact of the early environment on the long term health of ART offspring. Study funding/competing interest(s)This work was funded by the National Council for Science and Technology of Mexico (CONACyT), an NIHR pre-doctoral fellowship (PCAF) to MM, the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility, the University of Manchester and Manchester University NHS Foundation Trust. None of the authors has any conflict of interest to declare.

ImportanceLimited knowledge exists on the effects of SARS-CoV-2 infection after embryo transfer, despite an increasing number of studies exploring the impact of previous SARS-CoV-2 infection on IVF outcomes. ObjectiveThis prospective cohort study aimed to assess the influence of SARS-CoV-2 infection at various time stages after embryo transfer on pregnancy outcomes in patients undergoing conventional in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI) treatment. DesignThe study was conducted at a single public IVF center in China. SettingThis was a population-based prospective cohort study. ParticipantsFemale patients aged 20 to 39 years, with a body mass index (BMI) between 18 and 30 kg/m2, undergoing IVF/ICSI treatment, were enrolled from September 2022 to December 2022, with follow-up until March 2023. ExposureThe pregnancy outcome of patients was compared between those SARS-CoV-2-infected after embryo transfer and those noninfected during the follow-up period. Main Outcomes and MeasuresThe pregnancy outcomes included biochemical pregnancy rate, implantation rate, clinical pregnancy rate, and early miscarriage rate. ResultsA total of 857 female patients undergoing IVF/ICSI treatment were included in the analysis. We observed the incidence of SARS-CoV-2 infection within 10 weeks after embryo transfer. The biochemical pregnancy rate and implantation rate were lower in the infected group than the uninfected group (58.1% vs 65.9%; 36.6% vs 44.0%, respectively), but no statistically significant. Although, the clinical pregnancy rate was significant lower in the infection group when compared with the uninfected group (49.1%vs 58.2%, p < 0.05), after adjustment for confounders, this increased risk was no longer significant between the two groups (adjusted OR, 0.736, 95% CI, 0.518-1.046). With continued follow-up, a slightly higher risk of early miscarriage in the infected group compared to the uninfected group (9.3% vs 8.8%), but it was not significant (adjusted OR, 0.907, 95% CI, 0.414-1.986). Conclusions and RelevanceThe studys findings suggested that SARS-CoV-2 infection within 10 weeks after embryo transfer may have not significantly affect pregnancy outcomes. This evidence allays concerns and provides valuable insights for assisted reproduction practices. Key pointsO_ST_ABSQuestionC_ST_ABSDid the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after embryo transfer affect pregnancy outcomes? FindingsIn this prospective cohort study involving 857 patients, we made a pioneering discovery that SARS-CoV-2 infection following embryo transfer did not exhibit adverse impact on the biochemical pregnancy rate, embryo implantation rate, clinical pregnancy rate, and early miscarriage rate. MeaningThe evidence from this study alleviates existing concerns and offers new insights into the actual risk of SARS-CoV-2 infection after embryo transfer in assisted reproduction.

ObjectivesTo determine who spends more energy over a lifetime on maintaining their reproductive potential: men or women? DesignAs a model and energetic equivalent, we set the mass of gametes supported over time from birth until exhaustion of fertility. We calculated gender-specific dynamics of gamete pool mass over time. To this purpose we collated data from existing literature, accounting for gamete volume over stages of development, time in each stage, mass density, and count. Our model generates the integral, or area under the curve (AUC) of the gamete pool mass over a lifetime as a proxy to energetic requirements. Main outcome measuresThe area under gamete mass curve over a lifetime in men and women. ResultsThe number of gametes over a lifetime is 600,000 in women and close to 1 trillion in men. Accounting for mass and time, women invest approximately 100 gram*days in maintaining the female oocyte pool. Women reach 50% of lifetime AUC by age 10, and 90% by age 25. Men invest approximately 30 Kg*days over a lifetime (300-fold more), reaching 50% of lifetime AUC at age 37 and 90% at age 62 years old. ConclusionsThe study quantifies for the first time the area under gamete mass in men and women through a nuanced calculation accounting for all components of post-natal gamete dynamics. We found a 300-fold excess is supported male gamete mass over a lifetime (100g*days vs. 30 Kg*days in females vs. males, respectively). Our methodology offers a framework for assessing other components of the reproductive system in a similar quantitative manner.

BackgroundPolycystic ovary syndrome is one of the most frequent endocrinological problems causing infertility in women worldwide. The main problem in these women is hyperandrogenism and/or chronic oligo/anovulation, which leads to infertility. In this systematic review and meta-analysis, we aimed to investigate the efficacy of a relatively new drug for ovulation induction, letrozole, by comparing it to the first line of treatment for ovulation induction, clomiphene citrate. MethodsA literary search was conducted in three databases and included randomized clinical trials comparing letrozole and clomiphene citrate for ovulation induction for women with polycystic ovary syndrome. The diagnosis of polycystic ovary syndrome was determined according to the Rotterdam criteria. We pooled data using a random-effects model. ResultsOur search provided a total of 1,994 articles, of which we included 25 studies. In the letrozole group, endometrial thickness was significantly higher (Mean Difference=1.70, Confidence Interval: 0.55-2.86; Heterogeinity: I2=97%, p-value=0.008); odds for ovulation (Odds Ratio=1.8, Confidence Interval: 1.21-2.69; Heterogeinity: I2=51%, p-value=0.010) and pregnancy (Odds Ratio=1.96, Confidence Interval: 1.37-2.81; Heterogeinity: I2=32%, p- value=0.002) were significantly higher; the resistance index of subendometrial arteries was significantly lower (Mean Difference=-0.15, Confidence Interval: -0.27- -0.04; Heterogeneity: I2=92%, p-value=0.030). ConclusionWomen with polycystic ovary syndrome treated with letrozole for ovulation induction had higher ovulation and pregnancy rates, their endometrium became thicker, the resistance index of subendometrial arteries was lower. The lower resistance index of the subendometrial arteries can improve intrauterine circulation, which may provide better circumstances for embryo implantation and development.

Background: Male factor infertility is present in around 40% of couples utilising assisted reproductive technology (ART). However, it is unclear how specific causes of male infertility impact the chance of successful ART treatment, with most research either treating male infertility as single diagnostic group or being isolated smaller-scale studies focused on the treatment and outcomes of specific diagnoses. Objective: To study the impact of eleven specific aetiologies of male infertility on the chance of a clinical pregnancy in couples with known causes of male or female infertility following their first ART cycle. Material and methods: Population-based (initiated ART in Australia and New Zealand in 2020- 2022) cohort study assessing the impact of eleven male infertility diagnoses (idiopathic, Klinefelter syndrome, Y chromosome microdeletions, testis damage from cancer, testis damage from other causes, gonadotropin deficiency, congenital absence of the vas deferens/cystic fibrosis (CBAVD), other obstruction disorder, erectile dysfunction, and ejaculatory disorder) on the chance of a clinical pregnancy following a couple's first complete ART cycle (all fresh and frozen-thawed embryo transfers arising from one episode of ovarian stimulation). Adjusted risk ratios comparing a couples undergoing ART solely for treatment of male infertility with couples undergoing ART solely for treatment of tubal disease were calculated for the chance of a clinical pregnancy following a complete ART cycle and following an attempted fertilisation procedure. Results: A total of 39,053 couples were included, with male infertility present in 42.7% of cases, and the only cause of infertility in just under half of these cases. In more than three-quarters of male infertility cases the cause of infertility was unknown (idiopathic) or undiagnosed. Most couples undertaking ART for treatment of male infertility can expect similar success rates to couples seeking treatment for good prognosis female infertility diagnoses. However, those with Klinefelter syndrome and Y chromosome microdeletions had a 59.5% (aRR: 40.5% [95% CI: 16.9%-64.1%]) and 28.9% (aRR: 71.1% [95% CI: 45.5%-96.7%]) lower chance of a clinical pregnancy per initiated stimulation cycle compared to those with tubal disease as the only source of infertility. However, there was no difference once sperm was retrieved compared to other diagnoses tending to require surgical sperm retrieval, use frozen oocytes and necessitating ICSI. Discussion and Conclusion: In this population-based study most couples undergoing ART because of male infertility had similar success rates to those undergoing ART for treatment of female tubal disease, except for patients with Klinefelter syndrome and Y chromosome microdeletion who had approximately half and three-quarters the chance of a clinical pregnancy due to failed sperm retrieval/survival, but no difference (accounting for the use of surgical sperm, ICSI and potentially frozen oocytes) in outcomes once sperm were available. While these finding are reassuring for most men presenting to an ART clinic with male infertility, with more than three-quarters of male infertility cases reported as being idiopathic, there is an urgent need for greater research on the causes, diagnosis and implications of male infertility.

ObjectiveTo evaluate whether hyaluronan-enriched transfer medium improves live birth rates in biopsied euploid blastocyst transfers and to examine the role of zona pellucida disruption in mediating this effect. DesignRetrospective cohort study. ParticipantsA total of 1,221 single frozen euploid blastocyst transfers performed between January 2011 and December 2024. InterventionEmbryo transfer using hyaluronan-enriched transfer medium compared with standard zwitterionic-buffered transfer medium. All embryos underwent trophectoderm biopsy resulting in zona pellucida disruption. Main Outcome MeasuresLive birth rate. Secondary outcomes included biochemical pregnancy and clinical pregnancy rates. ResultsHyaluronan-enriched transfer medium was associated with significantly higher live birth rates compared with standard medium (59.1% vs. 43.2%; absolute difference 15.9%, 95% confidence interval 10.3%-21.5%; relative risk 1.37, 95% confidence interval 1.22-1.54; P < 0.001). Clinical pregnancy and biochemical pregnancy rates were also significantly higher in the hyaluronan group (P < 0.001 for both comparisons). Sensitivity analysis restricted to first transfers per patient (n = 715) confirmed persistence of the live birth benefit (61.2% vs. 47.1%; absolute difference 14.1%, 95% confidence interval 6.9%-21.3%; relative risk 1.30, 95% confidence interval 1.13-1.49; P < 0.001). Maternal age was comparable between groups. ConclusionUse of hyaluronan-enriched transfer medium is associated with a clinically meaningful increase in live birth rates in biopsied euploid blastocyst transfers. Zona pellucida disruption created during trophectoderm biopsy may facilitate enhanced embryo-endometrial interaction, improving implantation efficiency.

IntroductionPregnancy loss occurs in 25% of all pregnancies. While 50-60% of the pregnancy losses are due to fetal chromosomal and structural abnormalities, incompatible with life, some of the remaining euploid pregnancy losses are likely caused by maternal conditions. At least 2% of all women suffer [&ge;]3 consecutive pregnancy losses, defined as recurrent pregnancy loss (RPL). A hyperactive maternal immune system has been suspected to be responsible for rejection of healthy fetuses and be the underlying cause for unexplained recurrent pregnancy loss. However, very sparsely evidence-based treatment is available. The anti-inflammatory immune modulatory effects of hydroxychloroquine (HCQ) and casuistic reports of live births by women with previous RPL after HCQ-treatment, have given rise to several hypothesizes for HCQs impact on RPL. In this multicenter randomized, double-blinded, placebo-controlled trial (db-RCT) we aim to uncover whether HCQ can increase the live birth rate (LBR) in women with RPL, thus laying the foundation for an evidence-based treatment to make a real difference for a much-overlooked group of patients. Methods and analysisWe will assess HCQ treatment in women with a minimum of four consecutive unexplained pregnancy losses (or three pregnancy losses including a second trimester pregnancy loss), and monitor a potential difference in LBR among women allocated treatment with HCQ and placebo, without exclusion of any included patients (Intention to treat analysis) and with exclusion of those with ectopic pregnancy, pregnancy loss due to chromosome abnormalities, neglect of treatment, induced abortion or those who withdraw from the treatment earlier than the protocol dictates (per protocol analysis). Secondary outcome measures include comparison of the rate of perinatal complications in the intervention group to the placebo group and examination of inflammatory and immunological mechanisms in peripheral blood. We aim to include 186 patients in the db-RCT. In addition, we will in a sub-study with 10 women monitor metabolic activity measured by ultra-low dose FDG-PET/CT scans before and during pregnancy. Metabolic activity correlates with grade of inflammation and will be measured in the uterus and immune-related tissue such as bone marrow, spleen, thymus, and lymph nodes. Ethics and DisseminationThe db-RCT including the PET/CT sub-study has been approved by The Regional Committee on Health Research Ethics, The Danish Medicines Agency, The Danish Data Protection Agency, and transferred to The Center for Data handling Capital Region of Denmark in May 2024. The trial conforms to good clinical practice guidelines (GCP) and has been registered at ClinicalTrials.gov (ClinicalTrials.gov ID NCT03305263). Findings will be disseminated through peer-reviewed journals and at professional conferences. Strengths and limitations of this studyO_LIAssessment of HCQ treatment in women with RPL to potentially increase LBR. C_LIO_LIAssessment of the rate of perinatal outcomes associability with HCQ treatment. C_LIO_LIRPL trials so far have struggled to obtain pregnancy tissue from PL and if obtained been severely contaminated with maternal tissue. We will use circulating cell-free fetal DNA (cffDNA), isolated from maternal peripheral blood for fetal genetic diagnostics as we demonstrated feasible in our recent Lancet publication. C_LIO_LINon-invasive monitoring of inflammation in the uterus before and during pregnancy by the means of ultra-low dose PET/CT scans. C_LI

BACKGROUNDMaternal-embryonic signalling involves intense and complex molecular exchange between the maternal reproductive system and the early embryo. This interaction begins after fertilisation but is poorly understood before implantation. EARLY-PREG is structured as a bidirectional prospective-retrospective preconception open cohort designed to characterise time-dependent molecular signatures of maternal-embryonic communication during the earliest stage of pregnancy. The cohort supports a dynamic and structured longitudinal biobank of maternal biological fluids, cells and tissues-derived samples collected during the peri-implantation window. STUDY DESIGNParticipants were recruited in three rolling waves from 2017 to 2024 so far, in Concepcion, Chile. Healthy women trying to conceive were enrolled and completed a survey with health, lifestyle, and sociodemographic data. Menstrual cycles were prospectively monitored with ovulation and fertile window ascertainment (ultrasound, fertility monitor, and/or LH strips) and followed for a maximum of six consecutive cycles or until implantation of a viable embryo exposure occurred. Each cycle was synchronised within a longitudinal repeated-measures design incorporating systematic day-by-day sampling anchored to ovulation (day 0). Biological samples include cervicovaginal fluid (CVF), urine, saliva, and blood. In addition, cervical brushings were collected during the peri-implantation window (days 12-14 post-ovulation) and on day 21 post-ovulation. Retrospective temporal alignment of ovulation and defined physiological windows was performed using hormonal curves (LH, oestradiol, progesterone and beta-hCG) measured in stored blood and urine samples. RESULTS TO DATEAt present, 1,183 women have been contacted, of whom 223 met all eligibility criteria. Among participants trying to conceive, 129 completed at least one full longitudinal repeated-measures cycle; 35 achieved full-term pregnancies and 17 experienced early pregnancy loss (ELP). In addition, 40 abstinent and 5 sterilised women completed the protocol. To date, 292 menstrual cycles have been fully documented and sampled. 52 cycles correspond to conception cycles and 240 cycles to the absence of embryo implantation. Among the latter, 31 cycles were classified as non-conception counterfactual cycles and 209 to non-counterfactual cycles. The biorepository encompasses maternal biological samples collected during the first 2 weeks after ovulation across all documented cycles. Biospecimen collection compliance exceeded predefined protocol thresholds for most sample types. CURRENT AND FUTURE DIRECTIONSThe cohort currently supports longitudinal proteomic analyses within a within-individual counterfactual framework, comparing the exposure to a viable embryo implantation (conception cycle) with the absence of embryo implantation (non-conception cycle) to characterise time-dependent molecular signatures of early pregnancy. Additional outcomes of the cohort include implantation failure and early pregnancy loss. A fourth rolling recruitment wave is planned for 2026 to characterise time-dependent immunophenotypic variation in maternal peripheral blood mononuclear cells (PBMCs) during early pregnancy stages. STUDY FUNDINGThis work forms part of the EARLY-PREG preconception open cohort and has been supported by research grants awarded by Fundacion de Investigacion San Ramon (FISAR), Chile. The pilot study and first recruitment wave were supported by grants #MEL109112011 and #MEL109112011R4 awarded to E.S.K., C.V., and J.F.S. The second recruitment wave was supported by grants #MEL109112011R5 and #MEL131032017R1 awarded to E.S.K. The third wave was supported by grant #MEL205062018 awarded to E.S.K. and M.H. Current funding for the fourth recruitment wave and mass spectrometry research on maternal CVF is supported by grant REH042024-01 awarded to M.H., G.N., and E.S.K. TRIAL REGISTRATION NUMBERNCT07358026.

BackgroundIn vitro fertilization (IVF) has revolutionized reproductive medicine; however, IVF-conceived offspring exhibit a higher risk of various long-term health issues. The underlying mechanisms remain unclear. Long Interspersed Nuclear Element-1 (L1), a mobile genetic element sensitive to environmental stress, is a potential mediator. We hypothesized that the IVF procedure acts as an embryonic stress, altering L1 retrotransposition and contributing to genomic instability associated with disease risk. MethodsWhole blood from 33 IVF and 42 naturally conceived (NC) offspring were collected for deep sequencing. We quantified L1 genomic content and mapped novel L1 insertions and deletions with Bowtie2 and MELT, and further compared their frequencies and genomic distributions between the two groups. Gene-disease association analysis was performed on genes within 500kb of differential L1 sites. ResultsThe overall L1 content was significantly higher in IVF offspring compared to NC controls (P<0.05), a finding robustly confirmed in three sibling pairs from the same parents where the IVF children had higher L1 content than their NC siblings. We identified 11 specific genomic loci with significantly different L1 insertion frequencies and 14 loci with different deletion frequencies between IVF and NC offspring. Notably, these differential sites were often located near genes significantly associated with metabolic, cardiovascular, neuropsychiatric, and neoplastic diseases, conditions previously linked to IVF conception. ConclusionOur findings demonstrate that IVF conception is associated with increased L1 content and altered genomic distribution of L1 elements in offspring. The disease-related genes near these aberrant L1 sites provide a plausible genomic link between IVF-associated embryonic stress and the increased risk of specific diseases in the IVF population. This study offers novel insights into the molecular safety of ART.