Human Reproduction

Study question Do shortened blastocyst vitrification and warming protocols provide comparable live birth rates (LBR) and obstetrical and perinatal outcomes to traditional vitrification and warming protocols? Summary answer Shortened vitrification and warming protocols provide comparable LBR, obstetric and perinatal outcomes to traditional protocols. Shortened vitrification coupled with traditional multi step warming benefitted women >35yrs. What is known already Embryo viability following cryopreservation is dependent on blastomere survival and functional integrity, both impacted by ice crystal formation and osmotic gradients. Recent innovations in cryopreservation challenge the need for stepwise dehydration and rehydration protocols. While one step ''fast'' blastocyst warming protocols seem to provide equivalent clinical outcomes to traditional ''slow'' protocols, fewer studies investigate whether blastocyst dehydration rates can be similarly increased. A thorough safety and effectiveness evaluation remains necessary for both treatment success and offspring health. Study design, size, duration Three clinics within a network participated in this retrospective consecutive cohort study, with cycle data collected for 3603 warmed blastocysts resulting in 3168 frozen blastocyst transfers in 2170 patients between 2023 and 2025. We modelled the relationship between ''fast'' versus ''slow'' protocols and outcomes with Generalized Additive Models, and linear and logistic regressions where appropriate. Two tailed chi square with Yates correction was used to examine pregnancy loss and obstetrical and perinatal outcomes; p<0.05 was considered significant. Participants/materials, setting, methods All cycles were with the patients own gametes and without preimplantation genetic testing. All blastocysts were collapsed before vitrification, which was either a traditional ''slow'' protocol (Sydney IVF, Cook Medical) or an amended 2.5 minute ''fast'' one (SAGETM, CooperSurgical). Warming was either a traditional ''slow'' multi step protocol or a one step 1min ''fast'' protocol in 1.0M sucrose (both SAGETM, CooperSurgical). Main results and the role of chance The overall LBR was 33.1% (1050/3168) with female age 35.2{+/-}4.5 (range:22-46). Three groups were established for analysis from survival to live birth ''traditional vitrification/multi step warming (S/S, n=751 blastocysts warmed) short vitrification-multi step warming (F/S, n=991), and short vitrification/one-step warming (F/F, n=1593). S/S was set as the control group for statistical purposes. Overall, all reproductive outcomes, including live birth rates, as well as obstetric and perinatal outcomes, were comparable across all groups (all p>0.05). Importantly, women 35yrs or older at vitrification (n=1715 transfers) profited from a F/S strategy, which provided a significant increase in live birth rates (OR:1.42 [1.02-1.98] p=0.038) compared to S/S. The same improved live birth following a F/S strategy were also seen in embryos of lower quality (OR:1.78 [1.12-2.83] p=0.015), suggesting of a protective effect of this cryopreservation strategy on the developmental competence of impaired germplasm. Limitations, reasons for caution Factors affecting the results may be unaccounted for by the study retrospective nature. Wider implication of the findings Overall, shortened, ''faster'' vitrification and warming protocols provide comparable reproductive outcomes to traditional ones. The combination of shorter exposure to cryoprotectant (CPA) during vitrification and stepwise osmotic gradient during warming provided significant clinical benefits specifically to patients >35 and lower quality embryos, pointing to the possibility of adapting vitrification protocols to specific patients populations and optimizing their clinical outcomes.

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.

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

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 determine whether endometrial scratch induces differences in transcriptomic profiles or epithelial cell ciliation in the endometrium at the window of implantation that associate with live birth following IVF. DesignSecondary analysis of 50 matched endometrial biopsies collected within a randomised controlled trial evaluating the clinical effectiveness of endometrial scratch before first-time IVF. SettingEndometrial biopsy samples were obtained from women attending the Jessop Wing of Sheffield Teaching Hospitals. Population or SampleWomen undergoing first-time IVF who received an endometrial scratch in the preceding cycle at the window of implantation (6-10 days after LH surge). MethodsEndometrial biopsies were molecularly dated using transcriptomic menstrual-cycle staging algorithms. Bulk RNA sequencing was analysed using DESeq2 with FDR correction, and principal component analysis (PCA) assessed clustering patterns. Epithelial cell ciliation was quantified using immunohistochemistry and an automated Python-based image analysis pipeline. Main Outcome MeasuresDifferential endometrial gene expression between women with and without live birth after IVF; percentage coverage of ciliated epithelial cells in luminal and glandular regions. ResultsTranscriptomic dating confirmed no differences in menstrual-cycle timing between live-birth and no-live-birth groups. No significant differential gene expression was detected (log2FC >2, FDR <0.05), and PCA showed no clustering by pregnancy outcome. Ciliation coverage did not differ between outcome groups or between glandular and luminal surfaces. ConclusionsWhen implantation timing is precisely defined, endometrial scratch does not produce detectable transcriptomic changes or alterations in epithelial ciliation that distinguish women who achieve live birth after IVF. FundingWellbeing of Women RG2147; Wessex Medical Research; Rosetrees Trust (PGS23/100171).

Human reproduction is inherently inefficient1 and 1 in 6 people worldwide suffer infertility. In vitro fertilisation (IVF) can help some couples conceive, but only ~30% of cycles are successful. One factor affecting IVF efficacy is mitotic-origin (mosaic) aneuploidy in which embryos contain a mixture of cells with different numbers of chromosomes2. We previously showed that chromosome segregation error phenotypes are frequent in the first mitotic division of the human embryo3. However, the cause of these errors and impact on daughter cell karyotype is unknown. Here, using live chromosome imaging and next generation sequencing we show that activation of the microtubule depolymerase KIF2C reduces chromosome segregation errors and mitotic-origin aneuploidy at the 2-cell stage. The number of first divisions that show alternative cleavage patterns (associated with failed embryo development in IVF clinics) are also reduced with KIF2C activation. Our findings demonstrate that modulation of microtubule dynamics is a potential therapeutic route to improving human embryo quality and IVF outcomes.

Polycystic ovary syndrome is the most common endocrine condition in women and anovulatory cause of female infertility. While a pro-inflammatory cytokines and leukocyte bias in systemic circulation is well-documented in PCOS, it is not known how this inflammation extends to or affects the ovary. Additionally, the relationship between ovulation and inflammation in PCOS is not well-defined. We hypothesize that the ovarian follicular immune environment in PCOS is uniquely dysregulated, and that resolving anovulation through ovulation induction is not sufficient to alleviate this dysregulation. Using single-cell RNA and surface protein analysis of peripheral blood and follicular fluid from patients undergoing in vitro fertilization, we discovered that both control and PCOS follicles were immunologically distinct from circulation. At a systemic level, we find that ovulation induction in PCOS does not alleviate systemic inflammation. In contrast, while healthy control ovaries experienced acute immune-directed ovulatory signaling, PCOS ovarian follicles were deficient in key pro-ovulatory cell to cell communication, and displayed instead a chronic low-grade inflammatory state with fibrotic features. Taken together, a picture emerges where acute ovulation demonstrates a well-ordered series of follicle-specific immune information flows, which are disrupted and replaced by low grade chronic inflammation in the PCOS follicle.

Study questionHow are the levels of anti-Mullerian hormone and inflammatory cytokines influenced by superficial peritoneal endometriosis (SPE)? Summary answerFertility metrics (Endometriosis Fertility Index (EFI), and serum anti-Mullerian hormone (AMH) levels) are reduced in women with SPE. Simultaneously, inflammatory markers are elevated in the circulation and local pelvic peritoneal microenvironment, with distinct changes in each compartment. What is known alreadyBetween 25-40% of women with endometriosis experience infertility, though the mechanisms behind this are poorly understood. Ovarian endometriosis is known to decrease AMH levels and contribute to infertility, but little is known about SPE-associated infertility, and how the peritoneal microenvironment might play a role in infertility for women with SPE. Study design, size, durationVenous blood samples from women with suspected endometriosis were collected prior to diagnostic laparoscopy (n=105). Pelvic peritoneal fluid was also collected from a subset of the women (n=38). The Endometriosis Fertility Index (EFI) was calculated after surgery, and levels of AMH and inflammatory cytokines measured by ELISA or multiplex Luminex. Participants/materials, setting, methodsBased on their surgical findings, women were classified as no endometriosis observed (no endo; n=39), superficial peritoneal lesions only (SPE; n=43), or SPE with an ovarian endometrioma (SPE+OE; n=23). Women were further grouped by their use of hormone treatments to manage their endometriosis symptoms (no endo: no hormones n=14, hormones n=25; SPE: no hormones n=20, hormones n=23; SPE+OE: no hormones n=17, hormones n=6). Data are described as either mean {+/-} standard deviation, or median [interquartile range]. Main results and the role of chanceSPE+OE women were older (31.73{+/-}6.31) than SPE (27.77{+/-}6.14; p=0.04) and control women (27.65{+/-}5.81; p=0.02). Both SPE and SPE+OE groups had lower EFI scores compared to women with no endometriosis (no endo 9.41{+/-}0.50; SPE 8.63{+/-}1.11 p=0.04, SPE+OE 6.95{+/-}1.60 p<0.0001). Serum AMH levels were lower for SPE alone (p=0.009) and SPE+OE women (0.73ng/mL [0.32, 1.19], p=0.002) compared to women with no endometriosis (1.15ng/mL [0.75, 1.94]) when accounting for age. When also accounting for hormone use, women with SPE+OE had lower AMH levels compared to women with no endometriosis (p=0.02), while women with SPE alone did not (p=0.069). Moreover, women with SPE not using hormones had elevated serum IL-17 (4.45pg/mL [4.26, 4.88] vs 3.84pg/mL [3.54, 4.19], p=0.02) and TNF- compared to women with no endometriosis (4.28pg/mL, [3.37, 5.88] vs 1.99pg/mL, [1.49, 3.43], p=0.03), while pelvic peritoneal fluid levels of IL-23 were elevated in women with SPE not using hormones (212.4pg/mL, [184.0, 244.5] vs 121.3, [46.37, 147.60], p=004). These differences were not significant in women using hormones. Limitations, reasons for cautionDue to the limited sample size of women not using hormones, we were unable to determine if serum IL-17 or TNF-, or pelvic peritoneal IL-23 levels negatively correlated with AMH levels. Wider implications of the findingsWomen with SPE, with or without OE, have lower AMH levels - indicative of reduced ovarian reserve - compared to women without endometriosis. Among those with SPE, diminished AMH was associated with increased serum levels of IL-17 and TNF- and elevated IL-23 in the pelvic peritoneal fluid, suggesting compartment-specific inflammatory profiles. Notably, changes to circulating inflammatory cytokines were different when use of hormonal therapy was taken into account, highlighting such treatments may modulate inflammation linked to endometriosis. Taken together, our data support the need for further investigation into inflammation as a potential mechanism underlying infertility in women with SPE in the absence of OE. Study funding/competing interest(s)Deanery of Clinical Sciences Funding Challenge, University of Edinburgh awarded to MJG. Trial registration numberUniversity of Edinburgh Lothian Ethics Committee REC 20/LO/1298.

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%.

Progestin-IUD use was more frequent among IUA cases (29.9%) than polyp (3.4%) or infertile (11.1%) comparison groups. Compared to infertile comparators, any prior progestin-IUD use was independently associated with IUA (aOR 3.12; 95% CI 2.01, 4.85). There was a duration-response pattern: use of 5 years or less was modestly associated with IUA case status (aOR 1.99; 1.09, 3.64), whereas use >5 years conferred more than a seven-fold increase (aOR 7.26; 3.27, 16.11). The association persisted among surgically naive women (aOR 3.98; 2.44, 6.48) and was concentrated in those who were nulliparous, where use beyond five years conferred an approximately twelve-fold increase in odds (aOR 12.74; 5.25, 30.92). Progestin-IUD use was less frequent in polyp controls relative to IUA and infertile comparators, suggesting a possible role for progestin exposure in preventing endometrial polyp formation. The case control design does not allow for estimation of absolute risk for an individual and cannot inform causation. Further prospective studies are needed to better assess the relationship between progestin-IUD's, particularly when used beyond five years, and adverse fertility outcomes.

Human embryo implantation, occurring approximately one week after fertilization, remains poorly understood due to ethical and technical limitations of in vivo investigation. To overcome these barriers, and model this critical developmental event, encompassing peri- and early post-implantation stages, we used an in vitro embryo attachment model composed of donor-derived endometrial epithelial cells forming an open-faced endometrial layer (OFEL) and human stem cell-derived blastoids recapitulating human day 5 blastocysts in peri-implantation model. Following attachment, developmental progression was further investigated on laminin-coated substrates to capture early post-implantation dynamics. Despite its central role as the primary endocrine signal of early pregnancy, human chorionic gonadotropin (hCG) remains largely uncharacterized in this context. Here, we describe the transcriptomic profile of blastoid-endometrial co-cultures relative to OFEL alone, identifying CGA and CGB3/5/8 as among the most strongly upregulated genes following blastoid attachment to hormonally stimulated OFEL. Consistent with these findings, immunoassays and luteinizing hormone/choriogonadotropin receptor (LHCGR) activation assays of conditioned media confirmed the secretion of heterodimeric, biologically active hCG and its free subunits in co-cultures, but not in endometrial layers alone. Notably, the hyperglycosylated hCG heterodimer was the predominant isoform detected. Co-culture with the endometrial component significantly increased hCG secretion compared with blastoids cultured alone, an effect further enhanced by hormonal priming in the peri-implantation model. Collectively, these findings indicate that a hormonally primed endometrial environment not only promotes blastoid attachment but also amplifies embryonic hCG production and bioactivity, underscoring the importance of maternal endocrine cues in early embryo-endometrium communication. Furthermore, our peri- and early post-implantation models recapitulate key aspects of reciprocal endocrine signaling between embryonic and endometrial tissues, providing a tractable experimental framework to investigate embryo-endometrium crosstalk.

BackgroundCurrent IVF calculators provide either cumulative success probabilities, such as the CDC IVF Success Estimator [1] and OPIS calculators [2, 3, 4, 5], or stage-specific point estimates such as the Orchid Embryo Banking Calculator [6], but they do not quantify uncertainty and cannot incorporate patient-specific outcomes observed during treatment. ObjectiveTo develop a distribution-based framework that (i) produces full probability distributions at each IVF stage and (ii) allows downstream predictions to update when new stage outcomes are known. MethodsWe constructed a sequential probabilistic model using fresh, autologous IVF cycles from the Human Fertilisation and Embryology Authority (HFEA) registry (2017-2018) [7] for egg retrieval, maturity, and fertilization, and integrated published clinical studies totaling over 435,000 additional observations for blastocyst formation, euploidy, freeze/thaw survival, and live birth after euploid transfer. Models were validated using 70/30 train-test splits with out-of-sample performance metrics. Egg retrieval is modeled with zero-inflated negative binomial (ZINB) regression; downstream stages apply sequential binomial filters. A "known value selection" mechanism conditionally updates predictions when observed counts are entered. ResultsThe model generates full probability distributions at each stage of IVF rather than point estimates. Multi-cycle modeling enables comprehensive family planning assessments, while known value updating on combined distributions maintains cycle-specific biology rather than averaging outcomes. When observed values are entered, downstream distributions update accordingly, helping to guide clinical decisions. Out-of-sample validation demonstrates minimal overfitting with train-test R2 gaps under 0.007. Distribution evaluation confirms well-calibrated prediction intervals (50% coverage: 50.2%, 80% coverage: 79.2%, 95% coverage: 94.9%). The model is available as a web application at https://www.herasight.com/ivf-calculator. ConclusionsA distribution-based, sequential framework with conditional updating addresses key limitations of existing calculators by providing uncertainty-quantified, stage-aware predictions that adapt to patient-specific outcomes observed during care. Study Funding/Competing InterestsFunded by Herasight Inc. Authors are employees or consultants of Herasight.

Aneuploidy in human embryos is surprisingly prevalent and increases drastically with maternal age, resulting in miscarriages, infertility and birth defects. Frequent errors during the meiotic divisions cause this aneuploidy, while age-independent errors during the first cleavage divisions of the embryo also contribute. However, the underlying mechanisms are poorly understood, largely because these events have never been visualised in living human embryos. Here, using cell-permeable DNA dyes, we film chromosome segregation during the first and second mitotic cleavage divisions in human embryos from women undergoing assisted reproduction following ovarian stimulation. We show that the first mitotic division takes several hours to complete and is highly variable. Timings of key mitotic events were, however, largely consistent with clinical videos of embryos that gave rise to live births. Multipolar divisions and lagging chromosomes during anaphase were frequent with no maternal age association. In contrast, the second mitosis was shorter and underwent mostly bipolar divisions with no detectable lagging chromosomes. We propose that the first mitotic division in humans is a unique and highly error-prone event, which contributes to fetal aneuploidies.

Background: Literature on the role of thermal discomfort (heat- and cold-stress) on in-vitro fertilization (IVF) outcomes are scarce and inconclusive. This multi-center research examines association between heat stress and IVF treatment outcomes in Andhra Pradesh, which is prone to year around chronic heat stress. Methods: IVF data were abstracted from clinical chart review of all patients from three IVF from centers 2019 to 2023, which included time-stamped data on each IVF procedure, demographics and pre-existing comorbidities. Weather data were acquired from the National Climatic Data Center (NCDC). IVF outcomes were modelled with respect to time-lagged exposure to ambient temperature stratified by hyper- and hypo-thermic conditions using Poisson and logistic regressions depending on the scale of IVF outcomes adjusting for confounders. Results: Heat stress peaked in June, which corresponded with elevated number of spontaneous abortions/miscarriage (SAM). Under hypo- and hyper-thermic conditions a unit increase ambient temperature was associated with an 11% higher and an 8% lower number of oocytes retrieved, respectively. Adjusting for confounders, a 10 degree F increase in two-day lag heat stress was associated with a 30% higher odds of SAM (odds ratio ~ 1.03; 95% CI = 1.001 to 1.068; p-value < 0.043), and odds of PTB were 3 times higher when three day-lagged heat index (HI) was greater than 35 degree C (odds ratio 1.13 to 7.99; p < 0.05). Conclusion. Our findings warrant strategies to engage IVF patients in mitigating their exposure to thermal discomfort before and during the treatment.

IntroductionAccess to accurate, non-invasive diagnostics remains a critical unmet need in womens health. Menstrual effluence, containing endometrial tissue, immune cells, and microbial communities, represents a clinically relevant specimen for genomic and molecular pathology applications, yet has historically been underutilized due to concerns about sample integrity and variability. MethodsWe developed and validated a standardized, at-home tampon-based collection system designed to preserve nucleic acids at ambient temperature for clinical-grade analyses. 1,067 tampon samples from 328 participants underwent, RNA sequencing and metatranscriptomic profiling to assess specimen transcript integrity, diagnostic fidelity, and microbial composition over time. 12 patients were exome sequenced using matched menstrual effluence and whole blood to assess assay concordance between sample types. ResultsRNA extracted from menstrual effluence maintained stability for up to 14 days without refrigeration, achieving sufficient yield and quality for sequencing in >97% of samples. Variant detection via exome sequencing demonstrated 100% concordance among overlapping single nucleotide variants between menstrual fluid and matched venous blood, confirming clinical equivalency for genetic testing. Transcriptomic analyses revealed cycle-dependent variation in key reproductive and immune markers, while metatranscriptomic profiling identified shifts in microbial communities consistent with known reproductive tract dysbiosis. ConclusionsStandardized at-home collection of menstrual effluence provides a clinically actionable platform that supports remote specimen acquisition without compromising molecular assay fidelity, offering a scalable solution to improve access to carrier screening, reproductive health assessment, and infectious disease monitoring in clinical practice.

The recent availability of at-home menstrual cycle tracking technology has created opportunities for personalized assessment of reproductive health, alongside improved characterization of hormone patterns in women with and without reproductive disorders such as polyendocrine metabolic ovarian syndrome (PMOS), which affects approximately 10% of reproductive-age women. In this study, we leverage self-tracked urinary hormone data to develop an autoregressive Hidden Markov model (arHMM) that maps cycle days to physiologically meaningful phases based on hormone trajectories. By modeling day-to-day hormonal dynamics rather than absolute hormone levels, and allowing variable phase durations, this approach accommodates substantial variability in menstrual cycles, thereby enabling meaningful comparisons within and between individuals. Across more than 3800 cycles from over 1100 individuals, we find that arHMM-derived phases reproduce expected hormonal patterns within follicular, periovulatory, and luteal phases, and that phase-based timing for hormone testing outperforms conventional cycle day-based testing in capturing the luteinizing hormone surge and post-ovulatory progesterone rise, highlighting limitations of fixed-day clinical protocols. We identify phase-specific differences between healthy controls and individuals with self-reported PMOS, including lower luteinizing hormone in the periovulatory phase, and reduced luteal-phase progesterone levels in PMOS. Furthermore, features derived from arHMM phase assignments enable classification of PMOS status with ~78% accuracy, demonstrating the potential of this approach for non-invasive PMOS screening.

BackgroundThreatened miscarriage represents one of the most prevalent obstetric emergencies globally. Nevertheless, women experiencing first-trimester bleeding with a viable intrauterine pregnancy are predominantly advised based on empirical experience rather than on precisely quantified risks. Over the past forty years, numerous biochemical, ultrasound, and clinical predictors have been proposed. More recently, multivariable and machine-learning models have also been introduced. However, it remains uncertain which of these diagnostic tests genuinely contribute prognostic value and whether integrated modelling approaches significantly surpass routine assessment methods. MethodsWe conducted a comprehensive systematic review and a meta-analysis of prognostic accuracy, incorporating an evidence synthesis of multivariable and machine learning prediction models, focusing on women experiencing first-trimester threatened miscarriage with ultrasound-confirmed viable intrauterine pregnancy. Searches were performed across MEDLINE, Embase, CINAHL, CENTRAL, Web of Science, ClinicalTrials.gov, and WHO ICTRP from inception until March 2025, with no restrictions on language. Eligible studies encompassed prospective or nested cohort studies, or high-quality systematic reviews and meta-analyses, which reported first-trimester biochemical, ultrasound, clinical, or combined prediction models alongside subsequent pregnancy outcomes (including miscarriage versus ongoing pregnancy or live birth). Data were systematically extracted into a comprehensive template and synthesized following the principles outlined in PRISMA 2020/PRISMA-DTA, TRIPOD, CHARMS, QUADAS-2, and PROBAST. For thresholds reported in four or more studies, comparable in nature, we employed random-effects bivariate or HSROC models; when fewer such studies were available, the accuracy data were summarized narratively. FindingsTen studies met the inclusion criteria: six primary prospective cohorts and four evidence-synthesis or meta-analytic papers from early pregnancy and emergency settings. Among the primary cohorts, miscarriage risks consistently ranged from 15% to 25% in women with threatened miscarriage and an initially viable intrauterine pregnancy, confirming this as a genuinely high-risk state rather than a benign variation of normal pregnancy. Serum progesterone showed high specificity (>80-90%) but modest pooled sensitivity ([~]30%), indicating that very low values strongly "rule in" risk but fail to identify many women who miscarry. In contrast, CA-125 demonstrated near single-test performance in two large meta-analyses (pooled sensitivity and specificity both {approx}approximately 90-95%, AUC {approx}approximately 0.95), and in one cohort, a cut-off around 31 IU/mL yielded sensitivity over 95% and specificity of 100%. Ultrasound viability parameters, particularly fetal heart rate, crown-rump length, and gestational sac morphology, were the most consistently informative imaging predictors, with specificities often [&ge;]85-90% and good to fair discrimination but limited sensitivity and varied cut-offs. Only two recent cohorts developed formal multivariable models: logistic regression models combining maternal factors, progesterone, and ultrasound features achieved AUCs in the high 8 to about 0.9 range, while a single random forest model reached an apparent AUC of approximately 97, with very high negative predictive value on internal testing validation. All models, however, were from single centers, based on modest event numbers, had moderate-to-high risk of bias, and lacked external validation. Common threats to validity across the literature included non-consecutive recruitment, post-hoc threshold selection, incomplete follow-up, and insufficient reporting to reconstruct 2x2 data. InterpretationFor women experiencing threatened miscarriage in the first trimester with a viable intrauterine pregnancy, substantial prognostic indicators are already available; however, these indicators tend to be fragmented, lack sufficient statistical power, and are seldom translated into practical tools. Ultrasound viability parameters, combined with a limited selection of biochemical markers, particularly CA-125 and progesterone, can effectively stratify risk and facilitate more accurate, probabilistic counseling. Nonetheless, no individual test or model currently satisfies the evidentiary standards necessary for inclusion in clinical guidelines implementation. The priority within the field should now transition from the discovery of increasingly isolated predictors to the cultivation of extensive, prospectively registered, multicentre cohorts characterized by harmonized definitions, prespecified multimodal predictor sets, rigorous contemporary modelling techniques, and comprehensive TRIPOD-compliant reporting alongside external validation. This synthesis delineates the most robust existing indicators, identifies the methodological deficiencies that compromise current models, and offers a concrete roadmap for the development of truly reliable prognostic tools intended for the millions of women worldwide who face threatened miscarriage annually.

Preimplantation genetic testing for aneuploidy (PGT-A) aims to improve IVF outcomes by identifying embryos with lethal chromosomal abnormalities leading to impaired embryonic development and pregnancy loss, particularly with advancing maternal age. The adoption of next-generation sequencing (NGS) has enabled the detection of subtler chromosomal variations of uncertain clinical significance, such as intermediate copy number (ICN), leading to the classification of putative mosaicism. To assess the clinical utility of mosaicism reporting in PGT, we conducted a large, multi-site, double-blinded, non-selection study across U.S. fertility clinics (Feb 2020-Oct 2022), analyzing 9,828 single embryo transfers (SETs) from 7,564 IVF cycles. The findings were independently validated using a separate dataset of 5,487 euploid SETs from European clinics (May 2022-March 2024), representing a distinct patient population with different clinical characteristics. This design allowed for ICN values to be unblinded post-transfer, enabling unbiased comparisons between embryos that, under a mosaic reporting framework, would have been labeled as mosaic or euploid. In both cohorts, the presence of ICN showed no significant impact on clinical outcomes when mosaicism status was blinded and evaluated alongside established clinical and embryological parameters. AUROC analysis revealed no meaningful difference in predictive performance between models with and without ICN data (AUC = 0.552 vs. 0.555; 0.569 vs. 0.578; all P > 0.05). Miscarriage, obstetric, and neonatal outcomes were also comparable. These findings strongly suggest that reporting putative mosaicism based on ICN lacks clinical utility and should not be used to inform embryo selection policies.

Objective To evaluate adherence to published American Society for Reproductive Medicine (ASRM) infertility evaluation and treatment recommendations among commercially insured infertility patients who subsequently underwent in vitro fertilization (IVF) and to assess whether observed care gaps support the need for a restorative reproductive medical framework. Methods A retrospective claims-based analysis was performed using MarketScan(R) Commercial Claims and Encounter Data between January 1, 2021, and December 31, 2024. Approximately five million commercially insured members were evaluated. Patients with infertility-related diagnoses who subsequently underwent IVF were identified. Claims were analyzed for evidence of diagnostic testing, medical treatment, or surgical intervention recommended by ASRM or AUA/ASRM guidance before IVF initiation. Cumulative adherence rates were assessed over nine months following initial infertility diagnosis. Results IVF initiation rose early and consistently exceeded completion of nearly all guideline-recommended evaluations and treatments. Observed care gaps ranged from approximately 13% to 78% for most recommended evaluations and treatments, with several measures demonstrating gaps exceeding 50 percentage points, suggesting substantial divergence between guideline recommendations and observed clinical practice. By 3 months, IVF initiation ranged from 28% to 39% across cohorts, while adherence to many recommended interventions remained low. Overall, by 9 months, IVF utilization commonly exceeded 70-85%, while many guideline-supported evaluations and treatments remained below 40% adherence, with several interventions remaining below 15%. These findings suggest substantial divergence between published infertility-care recommendations and observed pre-IVF practice patterns. From an RRM perspective, the gaps are clinically important because many recommended steps are directed toward identifying, correcting, restoring, or preserving reproductive function and anatomy before reproductive barriers are bypassed through IVF. Conclusions Many commercially insured infertility patients appeared to progress to IVF without documented evidence of diagnostic evaluation or therapeutic intervention recommended in ASRM and AUA/ASRM guidance. These findings raise important questions regarding the implementation of infertility guidelines before IVF and the extent to which patients receive meaningful opportunities for diagnosis-directed treatment of potentially reversible causes of infertility. The findings further suggest an important role for restorative reproductive medicine as a quality-of-care framework focused on comprehensive evaluation, correction of underlying dysfunction, preservation of reproductive anatomy and physiology, and optimization of patient-centered fertility care prior to attempts with assisted reproduction.

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.