Recommendations from the 2024 Australian evidence-based guideline for unexplained infertility: ADAPTE process from the ESHRE evidence-based guideline on unexplained infertility.

INTRODUCTION: The 2024 Australian evidence-based guideline for unexplained infertility provides clinicians with evidence-based recommendations for the optimal diagnostic workup for infertile couples to establish the diagnosis of unexplained infertility and optimal therapeutic approach to treat couples diagnosed with unexplained infertility in the Australian health care setting. The guideline recommendations were adapted for the Australian context from the rigorous, comprehensive European Society of Human Reproduction and Embryology (ESHRE) 2023 Evidence-based guideline: unexplained infertility, using the ADAPTE process and have been approved by the Australian National Health and Medical Research Council. MAIN RECOMMENDATIONS: The guideline includes 40 evidence-based recommendations, 21 practice points and three research recommendations addressing: definition - defining infertility and frequency of intercourse, infertility and age, female and male factor infertility; diagnosis - ovulation, ovarian reserve, tubal factor, uterine factor, laparoscopy, cervical/vaginal factor, male factor, additional testing for systemic conditions; and treatment - expectant management, active treatment, mechanical-surgical procedures, alternative therapeutic approaches, quality of life. CHANGES IN ASSESSMENT AND MANAGEMENT RESULTING FROM THE GUIDELINE: This guideline refines the definition of unexplained infertility and addresses basic diagnostic procedures for infertility assessment not considered in previous guidelines on unexplained infertility. For therapeutic approaches, consideration of evidence quality, efficacy, safety and, in the Australian setting, feasibility, acceptability, cost, implementation and ultimately recommendation strength were integrated across multidisciplinary expertise and consumer perspectives in adapting recommendations to the Australian context by using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) framework, which had not been used in past guidelines on unexplained infertility to formulate recommendations. The Australian process also included an established data integrity check to ensure evidence could be trusted to guide practice. Practice points were added and expanded to consider the Australian setting. No evidence-based recommendations were underpinned by high quality evidence, with most having low or very low quality evidence. In this context, research recommendations were made including those for the Australian context. The full guideline and technical report are publicly available online and can be accessed at https://www.monash.edu/medicine/mchri/infertility and are supported by extensive translation resources, including the free patient ASKFertility mobile application (https://www.askfertility.org/).

Development of an IVF prediction model for donor oocytes: a retrospective analysis of 10 877 embryo transfers.

STUDY QUESTION: Can we develop a prediction model for the chance of a live birth following the transfer of an embryo created using donated oocytes? SUMMARY ANSWER: Three primary models that included patient, past treatment, and cycle characteristics were developed using Australian data to predict the chance of a live birth following the transfer of an embryo created using donated oocytes; these models were well-calibrated to the population studied, achieved reasonable predictive power and generalizability when tested on New Zealand data. WHAT IS KNOWN ALREADY: Nearly 9% of ART embryo transfer cycles performed globally use embryos created using donated oocytes. This percentage rises to one-quarter and one-half in same-sex couples and women aged over 45 years, respectively. STUDY DESIGN, SIZE, DURATION: This study uses population-based Australian clinical registry data comprising 9384 embryo transfer cycles that occurred between 2015 and 2021 for model development, with an external validation cohort of 1493 New Zealand embryo transfer cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three prediction models were compared that incorporated patient characteristics, but differed in whether they considered use of prior autologous treatment factors and current treatment parameters. We internally validated the models on Australian data using grouped cross-validation and reported several measures of model discrimination and calibration. Variable importance was measured through calculating the change in predictive performance that resulted from variable permutation. The best-performing model was externally validated on data from New Zealand. MAIN RESULTS AND THE ROLE OF CHANCE: The best-performing model had an internal validation AUC-ROC of 0.60 and Brier score of 0.20, and external validation AUC-ROC of 0.61 and Brier score of 0.23. While these results indicate ∼15% less discriminatory ability compared to models assessed on an autologous cohort from the same population the performance of the models was clearly statistically significantly better than random, demonstrated generalizability, and was well-calibrated to the population studied. The most important variables for predicting the chance of a live birth were the oocyte donor age, the number of prior oocyte recipient embryo transfer cycles, whether the transferred embryo was cleavage or blastocyst stage and oocyte recipient age. Of lesser importance were the oocyte-recipient parity, whether donor or partner sperm was used, the number of prior autologous embryo transfer cycles and the number of embryos transferred. LIMITATIONS, REASONS FOR CAUTION: The models had relatively weak discrimination suggesting further features need to be added to improve their predictive power. Variation in donor oocyte cohorts across countries due to differences such as whether anonymous and compensated donation are allowed may necessitate the models be recalibrated prior to application in non-Australian cohorts. WIDER IMPLICATIONS OF THE FINDINGS: These results confirm the well-established importance of oocyte age and ART treatment history as the key prognostic factors in predicting treatment outcomes. One of the developed models has been incorporated into a consumer-facing website (YourIVFSuccess.com.au/Estimator) to allow patients to obtain personalized estimates of their chance of success using donor oocytes. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Australian government as part of the Medical Research Future Fund (MRFF) Emerging Priorities and Consumer Driven Research initiative: EPCD000007. L.R. declares personal consulting fees from Abbott and Merck, lecture fees from Abbott, receipt of an educational grant from Merck, past presidency of the Fertility Society of Australia & New Zealand and World Endometriosis Society and being a minor shareholder in Monash IVF Group (ASX:MVF). G.M.C. declares receipt of Australian government grant funding for the research study and the development and maintenance of the YourIVFSuccess website. O.F., J.N., and A.P. report no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Medicine in the marketplace: clinician and patient views on commercial influences on assisted reproductive technology.

RESEARCH QUESTION: What are the views and experiences of patient and expert stakeholders on the positive and negative impacts of commercial influences on the provision of assisted reproductive technology (ART) services, and what are their suggestions for governance reforms? DESIGN: Semi-structured interviews were conducted with 31 ART industry experts from across Australia and New Zealand and 25 patients undergoing ART from metropolitan and regional Australia, between September 2020 and September 2021. Data were analysed using thematic analysis. RESULTS: Expert and patient participants considered that commercial forces influence the provision of ART in a number of positive ways - increasing sustainability, ensuring consistency in standards and providing patients with greater choice. Participants also considered commercial forces to have a number of negative impacts, including increased costs to government and patients; the excessive use of interventions that lack sufficient evidence to be considered part of standard care; inadequately informed consent (particularly with regard to financial information); and threats to patient-provider relationships and patient-centred care. Participants varied in whether they believed that professional self-regulation is sufficient. While recognizing the benefits of commercial investment in healthcare, many considered that regulatory reforms, as well as organizational cultural initiatives, are needed as means to ensure the primacy of patient well-being. CONCLUSIONS: The views expressed in this study should be systematically and critically examined to derive insights into how best to govern ART. These insights may also inform the design and delivery of other types of healthcare that are provided in the private sector.

Cardiovascular disease hospitalizations among women who undergo fertility treatment.

RESEARCH QUESTION: Are women who receive fertility treatment at increased risk of cardiovascular disease (CVD) hospitalization compared with women who do not? DESIGN: A retrospective cohort study of all women registered for fertility treatment at Monash IVF between 1998 and 2014. This cohort was linked to the Victorian Admitted Episodes Dataset, which contains records of all hospital admissions in the Australian state of Victoria. Age- and Index of Relative Socioeconomic Disadvantage (IRSD)-adjusted relative risks of CVD hospitalization for women who did or did not undergo fertility treatment were determined using Poisson regression. Risks were calculated overall by CVD subtype and stratified by area-based social disadvantage using IRSD fifths, number of stimulated cycles and mean oocytes per cycle. RESULTS: Of 27,262 women registered for fertility treatment, 24,131 underwent treatment and 3131 did not. No significant difference was found in risk of CVD hospitalization between treated and untreated women overall (adjusted RR 0.93, 95% 0.82 to 1.05) or by CVD subtype. The admission risk for CVD was significantly lower in treated women who had a mean of fewer than five oocytes per cycle (adjusted RR 0.80, 95% CI 0.70 to 0.92) compared with untreated women. Treated women residing in areas within the second IRSD fifth were less likely to be hospitalized for CVD compared with untreated women (age-adjusted RR 0.66, 95% CI 0.49 to 0.89). CONCLUSIONS: Fertility treatment is not associated with increased risk of CVD hospitalization. Lower risk among some subgroups of treated women may be explained by social disadvantage.

World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonization Project: V. Physical examination standards in endometriosis research.

OBJECTIVE: The World Endometriosis Research Foundation established the Endometriosis Phenome and Biobanking Harmonisation Project (EPHect) to create standardized documentation tools (with common data elements) to facilitate the comparison and combination of data across different research sites and studies. In 2014, 4 data research standards were published: clinician-reported surgical data, patient-reported clinical data, and fluid and tissue biospecimen collection. Our current objective is to create an EPHect standard for the clinician-reported physical examination (EPHect-PE) for research studies. DESIGN: An international consortium involving 26 clinical and academic experts and patient partners from 11 countries representing 25 institutions and organizations. Two virtual workshops, followed by the development of the physical examination standards underwent multiple rounds of iterations and revisions. SUBJECTS: N/A MAIN OUTCOME MEASURE(S): N/A RESULT(S): The EPHect-PE tool provides standardized assessment of physical examination characteristics and pain phenotyping. Data elements involve examination of back and pelvic girdle; abdomen including allodynia and trigger points; vulva including provoked vestibulodynia; pelvic floor muscle tone and tenderness; tenderness on unidigital pelvic examination; presence of pelvic nodularity; uterine size and mobility; presence of adnexal masses; presence of incisional masses; speculum examination; tenderness and allodynia at an extra-pelvic site (e.g., forearm); and recording of anthropometrics. CONCLUSION(S): The EPHect-PE standards will facilitate the standardized documentation of the physical examination, including the assessment and documentation of examination phenotyping of endometriosis-associated pelvic pain.

Storage trends, usage and disposition outcomes following egg freezing.

RESEARCH QUESTION: What happens to eggs after egg freezing? DESIGN: A retrospective cohort study was performed spanning 2012-2022. Data were obtained from seven assisted reproductive technology clinics in Victoria, Australia. Aggregated, de-identified data were collected on cycles that resulted in egg freezing and the following outcomes, including treatment involving thawed eggs and disposition outcomes of surplus eggs. RESULTS: The number of patients with eggs in storage grew rapidly from 144 in 2012 to 2015 in 2022. In 2022, 73% of patients had stored their eggs for <5 years, 25% for 5-10 years, and 2% for ≥10 years. Most thaw cycles (600/645, 93%) involved eggs that had been frozen for <5 years, of which 47% had been frozen for <6 months. Overall, the live birth rate per initiated thaw cycle was 12%. Across the study period, 2800 eggs from 286 patients were either discarded, donated or exported. Of the 128 patients who discarded their eggs, 32% had stored their eggs for <5 years, 32% for 5-10 years and 36% for >10 years. Of the 23 patients who donated their eggs to someone else, all but four had stored their eggs for <5 years. No eggs were donated to research over the study period. CONCLUSIONS: This study shows that very few patients have made the decision to use or relinquish their eggs. Strategies may be needed to address the prolonged storage of surplus eggs, and ensure that patients are supported to make decisions regarding the fate of their eggs which align with their preferences and values.

Improved fertilization, degeneration, and embryo quality rates with PIEZO-intracytoplasmic sperm injection compared with conventional intracytoplasmic sperm injection: a sibling oocyte split multicenter trial.

OBJECTIVE: To investigate whether PIEZO-intracytoplasmic sperm injection (PIEZO-ICSI) increases the fertilization rate, decreases the degeneration rate, and increases the utilization rate per oocyte injected compared with conventional intracytoplasmic sperm injection (ICSI). DESIGN: Sibling oocyte split multicenter trial. SETTING: Fertility clinics. PATIENTS: Women with a diagnosis of infertility who used ICSI as their method of insemination and had ≥6 mature oocytes for injection. INTERVENTIONS: Participants had their mature oocyte cohort divided, where half were injected using conventional ICSI and the other half were injected using PIEZO-ICSI. For patients with an uneven oocyte number, the extra oocyte was injected using conventional ICSI. The injection technique used first was also randomized to ensure that there was no bias due to order of injection. MAIN OUTCOME MEASURE: The primary outcome measure was the fertilization rate after injection. RESULTS: A total of 108 patients underwent a sibling split use of conventional ICSI and PIEZO-ICSI. The fertilization rate was 71.6% in PIEZO-ICSI, which significantly increased compared with that in conventional ICSI 65.6%. In addition, the oocyte degeneration rate decreased in PIEZO-ICSI compared with that in conventional ICSI (6.3% vs. 12.1% respectively), and the blastocyst quality increased, as measured by the number of grade A and B quality blastocysts present on day 5 of development (33.3% vs. 27.5%). No significant differences in the aneuploidy or utilization rate, clinical pregnancy, or live birth outcome after single embryo transfer were noted between the two injection techniques. CONCLUSIONS: This trial supports the possibility that PIEZO-ICSI increases the fertilization rates, decreases the oocyte degeneration rates, and increases the blastocyst quality compared with conventional ICSI; however, it does not appear to influence the clinical pregnancy or live birth rate per transfer. CLINICIAN TRIAL REGISTRATION NUMBER: Australian and New Zealand Clinical Trial Registry ACTRN12620000407998.

Impact of male age on paternal aneuploidy: single-nucleotide polymorphism microarray outcomes following blastocyst biopsy.

RESEARCH QUESTION: Does advanced paternal age (APA; ≥40 years) contribute to a higher incidence of paternal origin aneuploidy in preimplantation embryos? DESIGN: This was a multicentre retrospective study of single-nucleotide polymorphism (SNP) microarray (Natera and Karyomapping) preimplantation genetic testing (PGT) outcomes of blastocyst-stage embryos. Whole-chromosome aneuploidy analysis was performed on 2409 embryos from 389 male patients undertaking 681 assisted reproductive technology (ART) cycles between 2012-2021. Segmental aneuploidy analysis was performed on 867 embryos from 140 men undertaking 242 ART cycles between 2016-2021. Embryos were grouped based on paternal age at sperm collection: <35, 35-39 and ≥40 years. Paternal and maternal origin aneuploidy rates were compared between groups using chi-squared and/or Fisher's exact tests. RESULTS: There was no significant difference across groups in paternal origin whole-chromosome aneuploidy rate, overall (P=0.7561) or when segregated by type (trisomy and monosomy: P=0.2235 and 0.8156) or complexity (single versus 2, 3 or ≥4 aneuploidies: P=0.9733, 0.7517, 0.669 and 0.1481). Conversely, maternal origin whole-chromosome aneuploidy rate differed across groups (P<0.0001) in alignment with differing mean maternal age (P<0.001). Paternal origin deletions were 2.9-fold higher than maternal origin deletions (P=0.0084), independent of age stratification. No significant difference in paternal origin deletions was observed with APA ≥40 compared with the younger age groups (4.8% versus 2.5% and 2.8%, P=0.5292). Individual chromosome aneuploidy rates were too low to perform statistical comparisons. CONCLUSIONS: No significant association was found between APA and the incidence of paternal origin aneuploidy in preimplantation embryos, irrespective of type or complexity. Thus, APA may not be an indication for PGT.

Endometriosis research priorities in Australia.

In Australia, endometriosis affects one in nine women and those assigned female at birth. Although endometriosis is more common than conditions such as diabetes, research funding for endometriosis research has historically been low in comparison. The National Action Plan for Endometriosis is an Australian Federal Government initiative designed to redress this imbalance, with a focus on research funding. Identification of research priorities, and subsequent funding allocation that is determined by consumer input is vital. An online survey focusing on Australia and New Zealand found that the highest general priorities were the treatment and management of endometriosis and its cause(s).

PIEZO-ICSI increases fertilization rates compared with conventional ICSI in patients with poor prognosis.

PURPOSE: Limited research has been published comparing PIEZO-ICSI with conventional ICSI. While positive effects have been documented in improving fertilization and degeneration, the outcomes in patients with previous poor results from conventional ICSI remain unclear. It is hypothesized that these patients may benefit the most from this form of insemination. METHODS: This retrospective paired within-patient cohort study investigated patients (n=72) undertaking PIEZO-ICSI after a previous conventional ICSI cycle resulted in poor outcomes (including low fertilization (<50%), high degeneration (>15%), and/or poor embryo development and utilization). Patients required at least five oocytes collected in both cycles and a period of less than 2 years between the cycles. The outcomes of both cycles were compared in respect to fertilization, degeneration, embryo utilization, and pregnancy rates. Further analyses were applied to patients <38 and ≥38 years of age, with <50% or ≥50% fertilization with conventional ICSI and with <20% or ≥20% utilization with conventional ICSI. RESULTS: PIEZO-ICSI resulted in significantly higher fertilization (61.9% vs 45.3%, P<0.0001) and lower degeneration (7.7% vs 18.2%, P=0.0001) when compared to the conventional ICSI cycles. The greatest benefit was seen in patients who had less than 50% fertilization or <20% utilization in their conventional ICSI cycle, with improvements in fertilization and degeneration rates resulting in a significantly higher number of embryos utilized (frozen or transferred) per cycle. CONCLUSIONS: PIEZO-ICSI improved fertilization, degeneration, and utilization rates in patients with previous poor outcomes from conventional ICSI. The number of embryos available for use per cycle was also increased. Further significant improvements were achieved in patients who exhibited poor fertilization (<50%) or low utilization (<20%) from conventional ICSI.

Long term all-cause and cardiovascular disease mortality among women who undergo fertility treatment.

OBJECTIVES: To compare age-adjusted all-cause and CVD mortality, relative to the general female population, for women registered for fertility treatment who received it and those who did not. DESIGN: Prospective cohort study; analysis of Monash IVF clinical registries data, 1975-2018, linked with National Death Index mortality data. PARTICIPANTS: All women who registered for fertility treatment at Monash IVF (Melbourne, Victoria), 1 January 1975 - 1 January 2014, followed until 31 December 2018. MAIN OUTCOME MEASURES: Standardised mortality ratios (SMRs) for all-cause and CVD mortality, for women who did or did not undergo fertility treatment; SMRs stratified by area-level socio-economic disadvantage (SEIFA Index of Relative Socioeconomic Disadvantage [IRSD]) and (for women who underwent treatment), by stimulated cycle number and mean oocytes/cycle categories. RESULTS: Of 44 149 women registered for fertility treatment, 33 520 underwent treatment (66.4%), 10 629 did not. After adjustment for age, both all-cause (SMR, 0.58; 95% CI, 0.54-0.62) and CVD mortality (SMR, 0.41; 95% CI, 0.32-0.53) were lower than for the general female population. All-cause mortality was similar for women registered with Monash IVF who did (SMR, 0.55; 95% CI, 0.50-0.60) or did not undergo fertility treatment (SMR, 0.63; 95% CI, 0.56-0.70). The SMR was lowest for both treated and untreated women in the fifth IRSD quintile (least disadvantage), but the difference was statistically significant only for untreated women. CVD mortality was lower for registered women who underwent fertility treatment (SMR, 0.29; 95% CI, 0.19-0.43) than for those who did not (SMR, 0.58; 95% CI, 0.42-0.81). CONCLUSION: Fertility treatment does not increase long term all-cause or CVD mortality risk. Lower mortality among women registered for fertility treatment probably reflected their lower socio-economic disadvantage.

Progesterone suppresses podocalyxin partly by up-regulating miR-145 and miR-199 in human endometrial epithelial cells to enhance receptivity in in vitro models.

Establishment of endometrial surface receptivity is crucial for the initiation of embryo implantation yet the molecular mechanisms are not well understood, especially in humans. We have recently discovered that podocalyxin (PODXL) is a critical negative regulator of human endometrial surface receptivity. PODXL is highly expressed in all epithelial and endothelial cells in the non-receptive endometrium, but down-regulated specifically in the luminal epithelium at receptivity. We have further shown that PODXL inhibits embryo implantation, and that PODXL down-regulation is essential for endometrial surface receptivity. Our previous study also indicated that progesterone down-regulates PODXL; however, the exact molecular regulations are unknown. Here, we investigated whether progesterone suppresses PODXL via microRNAs (miRNAs). We first bioinformatically predicted 13 miRNAs that may potentially target human PODXL, then experimentally determined whether any of these 13 miRNAs are altered in primary human endometrial epithelial cells (HEECs) by progesterone, and whether the identified miRNAs can affect PODXL expression in Ishikawa cells without progesterone and alter receptivity to embryo implantation. Progesterone significantly up-regulated miR-145 and miR-199 while suppressing PODXL in HEECs. When these two miRNAs were transfected into Ishikawa cells, both significantly down-regulated PODXL mRNA and protein in the absence of progesterone. Moreover, both miR-145 and miR-199 significantly enhanced receptivity of the Ishikawa monolayer to embryo implantation in in vitro models. This study thus provides in vitro evidence that PODXL is down-regulated by progesterone partly via miR-145 and miR-199 during the development of human endometrial epithelial receptivity. These results also reveal the likely importance of hormonal regulation of miRNAs for embryo implantation.

The effect of day 5 blastocyst assessment timing on live birth prediction and development of a prediction algorithm.

RESEARCH QUESTION: Does variation in day 5 assessment timing confound live birth prediction using snapshot blastocyst morphology and is it possible to develop a numerical prediction algorithm? DESIGN: Retrospective multicentre cohort study including 4851 autologous oocyte single day 5 fresh embryo transfers performed at 11 Monash IVF clinics between 2016 and 2020. Repeat cycles of the same patients were excluded to avoid clustering effects in regression analysis. RESULTS: Hours post insemination (HPI) at day 5 assessment (115.9 ± 2.6 h) significantly correlated with blastocyst developmental stage (r = 0.118, P < 0.001). Independent association (expressed as adjusted odds ratio [aOR] and 95% confidence interval [CI]) was identified between live birth and HPI (aOR 0.950, 95% CI 0.925-0.976, P < 0.001) after accounting for blastocyst morphology and a range of patient/cycle characteristics. Algorithms were constructed using four significant live birth predictors: HPI at day 5 assessment, blastocyst developmental stage (aOR 1.347, 95% CI 1.217-1.491, P < 0.001), morphological grade (aOR 1.314, 95% 1.197-1.443, P < 0.001) and maternal age (aOR 0.922, 95% CI 0.907-0.936, P < 0.001). Receiver operating characteristic (ROC) analysis showed consistent predicting performance of algorithms via five-fold cross-validation, with similar area under the ROC curve (AUC 0.718, 0.715, 0.720, 0.712, 0.726, P < 0.001, respectively, in development subsets; and AUC 0.718, 0.731, 0.709, 0.741, 0.684, P < 0.001, respectively, in validation subsets). A score (ranging from 0.1 to 4.7) calculator based on the final algorithm was subsequently created. CONCLUSIONS: Day 5 assessment timing is a confounding factor for live birth prediction using snapshot blastocyst morphology. A numerical algorithm incorporating day 5 assessment HPI, blastocyst morphology and maternal age can be developed for live birth prediction.

Reproductive function in men conceived with in vitro fertilization and intracytoplasmic sperm injection.

OBJECTIVE: To determine the semen quality and reproductive hormones of men conceived by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) compared with men conceived without assisted reproductive technology (ART). DESIGN: Cohort study. SETTING: IVF centers in Victoria and the Western Australian Raine Study. PATIENT(S): Men conceived with IVF/ICSI and men conceived without ART aged 18-25 years. INTERVENTION(S): Clinical review. MAIN OUTCOME MEASURE(S): The primary outcome was the prevalence of severe oligozoospermia (sperm concentration, <5 million/mL). The secondary outcomes were total sperm count, total and progressive motility, total motile count, normal morphology, and serum testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). RESULTS: There was no difference in the prevalence of severe oligozoospermia between 120 men conceived with IVF/ICSI and 356 men conceived without ART (9% vs. 5.3%). Men conceived with IVF/ICSI had similar sperm concentration, total sperm count, and total motile count but lower mean total (55.3% vs. 60.6%) and progressive (44.7% vs. 53.9%) sperm motility with higher mean normal morphology (8.5% vs. 5.4%). Differences in progressive motility (ß, -9.9; 95% confidence interval [CI], -16.7 - -3.0), normal morphology (ß, 4.3; 95% CI, 3.0-5.7), and proportion with abnormal morphology (adjusted odds ratios, 0.1; 95% CI, 0.04-0.5) remained significant after adjusting for confounders. Men conceived with IVF/ICSI had lower mean FSH (3.3 IU/L) and LH (3.9 IU/L) levels and higher mean testosterone levels (19.1 nmol/L) than controls (4.2 IU/L, 11.0 IU/L, and 16.8 nmol/L). CONCLUSION: This study of men conceived with IVF/ICSI found similar sperm output to men conceived without ART. Overall, the results are reassuring.

Podocalyxin inhibits human embryo implantation in vitro and luminal podocalyxin in putative receptive endometrium is associated with implantation failure in fertility treatment.

OBJECTIVE: To study whether endometrial epithelial podocalyxin (PCX) inhibits implantation of human embryos in vitro and in patients undergoing in vitro fertilization (IVF). DESIGN: We have recently identified PCX as a key negative regulator of endometrial epithelial receptivity. Podocalyxin is expressed in all epithelial cells in the nonreceptive endometrium, but is selectively downregulated in the luminal epithelium (LE) for receptivity. In the current study, we first investigated whether high levels of PCX in Ishikawa monolayer inhibit attachment and/or penetration of human blastocysts in in vitro models. We then examined PCX by immunohistochemistry in putative receptive endometrial tissues biopsied from 81 IVF patients who underwent frozen embryo transfer in the next natural cycle and retrospectively analyzed the association between PCX staining in LE and clinical pregnancy as a proxy of successful implantation. SETTING: RMIT University, Australia; Vrije Universiteit Brussel, Belgium. PATIENT(S): In vitro fertilization patients undergoing frozen/thawed embryo transfer. INTERVENTION(S): N/A. MAIN OUTCOME MEASURE(S): Endometrial epithelial PCX inhibits implantation of human embryos in vitro and in IVF patients. RESULT(S): High levels of PCX in Ishikawa monolayer significantly inhibited blastocyst attachment and penetration. Among the 81 putative receptive tissues, 73% were negative, but 27% were heterogeneously positive for PCX in LE. The clinical pregnancy rate was 53% in those with a PCX-negative LE but only 18% in those with a PCX-positive LE. If LE was positive for PCX, the odds ratio of no clinical pregnancy was 4.95 (95% Confidence interval, 1.48-14.63). CONCLUSION(S): Podocalyxin inhibits embryo implantation. Assessment of PCX may aid the evaluation and optimization of endometrial receptivity in fertility treatment.

Podocalyxin is a key negative regulator of human endometrial epithelial receptivity for embryo implantation.

STUDY QUESTION: How is endometrial epithelial receptivity, particularly adhesiveness, regulated at the luminal epithelial surface for embryo implantation in the human? SUMMARY ANSWER: Podocalyxin (PCX), a transmembrane protein, was identified as a key negative regulator of endometrial epithelial receptivity; specific downregulation of PCX in the luminal epithelium in the mid-secretory phase, likely mediated by progesterone, may act as a critical step in converting endometrial surface from a non-receptive to an implantation-permitting state. WHAT IS KNOWN ALREADY: The human endometrium must undergo major molecular and cellular changes to transform from a non-receptive to a receptive state to accommodate embryo implantation. However, the fundamental mechanisms governing receptivity, particularly at the luminal surface where the embryo first interacts with, are not well understood. A widely held view is that upregulation of adhesion-promoting molecules is important, but the details are not well characterized. STUDY DESIGN, SIZE, DURATION: This study first aimed to identify novel adhesion-related membrane proteins with potential roles in receptivity in primary human endometrial epithelial cells (HEECs). Further experiments were then conducted to determine candidates' in vivo expression pattern in the human endometrium across the menstrual cycle, regulation by progesterone using cell culture, and functional importance in receptivity using in vitro human embryo attachment and invasion models. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary HEECs (n = 9) were isolated from the proliferative phase endometrial tissue, combined into three pools, subjected to plasma membrane protein enrichment by ultracentrifugation followed by proteomics analysis, which led to the discovery of PCX as a novel candidate of interest. Immunohistochemical analysis determined the in vivo expression pattern and cellular localization of PCX in the human endometrium across the menstrual cycle (n = 23). To investigate whether PCX is regulated by progesterone, the master driver of endometrial differentiation, primary HEECs were treated in culture with estradiol and progesterone and analyzed by RT-PCR (n = 5) and western blot (n = 4). To demonstrate that PCX acts as a negative regulator of receptivity, PCX was overexpressed in Ishikawa cells (a receptive line) and the impact on receptivity was determined using in vitro attachment (n = 3-5) and invasion models (n = 4-6), in which an Ishikawa monolayer mimicked the endometrial surface and primary human trophoblast spheroids mimicked embryos. Mann-Whitney U-test and ANOVA analyses established statistical significance at *P ≤ 0.05 and **P ≤ 0.01. MAIN RESULTS AND THE ROLE OF CHANCE: PCX was expressed on the apical surface of all epithelial and endothelial cells in the non-receptive endometrium, but selectively downregulated in the luminal epithelium from the mid-secretory phase coinciding with the establishment of receptivity. Progesterone was confirmed to be able to suppress PCX in primary HEECs, suggesting this hormone likely mediates the downregulation of luminal PCX in vivo for receptivity. Overexpression of PCX in Ishikawa monolayer inhibited not only the attachment but also the penetration of human embryo surrogates, demonstrating that PCX acts as an important negative regulator of epithelial receptivity for implantation. LIMITATIONS, REASONS FOR CAUTION: Primary HEECs isolated from the human endometrial tissue contained a mixture of luminal and glandular epithelial cells, as further purification into subtypes was not possible due to the lack of specific markers. Future study would need to investigate how progesterone differentially regulates PCX in endometrial epithelial subtypes. In addition, this study used primary human trophoblast spheroids as human embryo mimics and Ishikawa as endometrial epithelial cells in functional models, future studies with human blastocysts and primary epithelial cells would further validate the findings. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study add important new knowledge to the understanding of human endometrial remodeling for receptivity. The identification of PCX as a negative regulator of epithelial receptivity and the knowledge that its specific downregulation in the luminal epithelium coincides with receptivity development may provide new avenues to assess endometrial receptivity and individualize endometrial preparation protocols in assisted reproductive technology (ART). The study also discovered PCX as progesterone target in HEECs, identifying a potentially useful functional biomarker to monitor progesterone action, such as in the optimization of progesterone type/dose/route of administration for luteal support. STUDY FUNDING/COMPETING INTEREST(S): Study funding was obtained from ESHRE, Monash IVF and NHMRC. LR reports potential conflict of interests (received grants from Ferring Australia; personal fees from Monash IVF Group and Ferring Australia; and non-financial support from Merck Serono, MSD, and Guerbet outside the submitted work. LR is also a minority shareholder and the Group Medical Director for Monash IVF Group, a provider of fertility preservation services). The remaining authors have no potential conflict of interest to declare. TRIAL REGISTRATION NUMBER: NA.

IVF add-ons in Australia and New Zealand: A systematic assessment of IVF clinic websites.

BACKGROUND: In vitro fertilisation (IVF) 'add-ons' are extra (non-essential) procedures, techniques or medicines, which usually claim to increase the chance of a successful IVF outcome. Use of IVF add-ons is believed to be widespread in many settings; however, information about add-on availability in Australasia is lacking. AIMS: To understand which add-ons are advertised on Australasian IVF clinic websites, and what is the evidence for their benefit. MATERIALS AND METHODS: A systematic assessment of website content was undertaken between December 2019-April 2020, capturing IVF add-ons advertised, including costs, claims of benefit, statements of risk or limitations, and evidence of effectiveness for improving live birth and pregnancy. A literature review assessed the strength and quality of evidence for each add-on. RESULTS: Of the 40 included IVF clinics websites, 31 (78%) listed one or more IVF add-ons. A total of 21 different add-ons or add-on groups were identified, the most common being preimplantation genetic testing for aneuploidies (offered by 63% of clinics), time-lapse systems (33%) and assisted hatching (28%). In most cases (77%), descriptions of the IVF add-ons were accompanied by claims of benefit. Most claims (90%) were not quantified and very few referenced scientific publications to support the claims (9.8%). None of the add-ons were supported by high-quality evidence of benefit for pregnancy or live birth rates. The cost of IVF add-ons varied from $0 to $3700 (AUD/NZD). CONCLUSIONS: There is widespread advertising of add-ons on IVF clinic websites, which report benefits for add-ons that are not supported by high-quality evidence.

Risk of foetal harm with letrozole use in fertility treatment: a systematic review and meta-analysis.

BACKGROUND: The aromatase inhibitor letrozole is increasingly recommended for ovulation induction, as it is more effective with fewer side-effects than other agents. But many clinicians are reluctant to use the drug for fertility treatment due to a strong-label warning against its use, which warns about congenital malformation risk to the foetus in women seeking pregnancy. OBJECTIVE AND RATIONALE: The aim of this study was to determine the risks of congenital malformations and pregnancy loss with letrozole compared with clomiphene primarily, and with other fertility drugs and natural conception. SEARCH METHODS: A systematic review and meta-analysis using PRISMA harms guidelines. We searched MEDLINE, EMBASE and other sources from inception until January 2020, with the MeSH words for 'letrozole' and pregnancy OR foetal/neonatal outcome. We included studies reported on congenital malformations in foetuses born to mothers conceived after fertility treatment, with letrozole versus clomiphene, placebo, gonadotrophins, metformin, natural conception or other agents, from randomised trials, comparative cohort studies and non-comparative observational cohorts. Quality of the studies was assessed using Cochrane risk of bias tool and Newcastle Ottawa Scale. The McMaster tool was used to assess the quality of reported harm for foetal congenital malformations in the studies. We compared the absolute risk of events using risk difference measures and pooled the findings using a fixed-effect model. We evaluated the statistical heterogeneity using forest plots and the I2 statistic and funnel plot to assess publication bias. We assessed the strength of evidence for congenital malformation and pregnancy loss as per the GRADE recommendations and with the Fragility index. OUTCOMES: We included 46 studies (18 randomised trials; 21 comparative cohorts; 7 non-comparative cohorts). Overall 2.15% (101/4697; 95% CI 1.7 to 2.5) of babies conceived on letrozole for fertility treatment had congenital foetal malformations. We did not observe a significant increase in congenital malformations with letrozole versus clomiphene in the randomised trials (risk difference (RD) 0.01, 95% CI -0.02, 0.03; I2 = 0%; 14 studies) and found a significant reduction in the cohort studies (RD -0.02, 95% CI -0.04, -0.01; I2 = 0%, 11 studies). The fragility index was 44% (7/16) (either an increase in the intervention arm or a decrease in control arm was needed to alter the results). The risks of pregnancy loss were not increased with letrozole versus clomiphene in the 14 randomised trials (RD -0.01, 95% CI -0.06, 0.04; I2 = 0%), and the risks were reduced in the six cohort studies (RD -0.09, 95% CI -0.17, -0.00; I2 = 68%). The GRADE quality of evidence was low to moderate for congenital malformations and pregnancy loss. We did not find any increased congenital malformation risk with letrozole versus gonadotrophins, natural conception or natural cycle ART, but the number of studies was small. WIDER IMPLICATIONS: There is no evidence that letrozole increases the risk of congenital foetal malformation or pregnancy loss compared with clomiphene, natural conception or other fertility agents, to warrant warning against its use. Given its therapeutic benefits and lack of evidence of harm to the foetus, clinicians should consider letrozole as first-line agent for ovulation induction.

Mitochondria-targeted therapeutics, MitoQ and BGP-15, reverse aging-associated meiotic spindle defects in mouse and human oocytes.

STUDY QUESTION: Do mitochondria-targeted therapies reverse ageing- and oxidative stress-induced spindle defects in oocytes from mice and humans? SUMMARY ANSWER: Exposure to MitoQ or BGP-15 during IVM protected against spindle and chromosomal defects in mouse oocytes exposed to oxidative stress or derived from reproductively aged mice whilst MitoQ promoted nuclear maturation and protected against chromosomal misalignments in human oocytes. WHAT IS KNOWN ALREADY: Spindle and chromosomal abnormalities in oocytes are more prevalent with maternal aging, increasing the risk of aneuploidy, miscarriage and genetic disorders such as Down's syndrome. The origin of compromised oocyte function may be founded in mitochondrial dysfunction and increased reactive oxygen species (ROS). STUDY DESIGN, SIZE, DURATION: Oocytes from young and old mice were treated with MitoQ and/or BGP-15 during IVM. To directly induce mitochondrial dysfunction, oocytes were treated with H2O2, and then treated the MitoQ and/or BGP-15. Immature human oocytes were cultured with or without MitoQ. Each experiment was repeated at least three times, and data were analyzed by unpaired-sample t-test or chi-square test. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature germinal vesicle (GV) stage oocytes from 1-, 12- and 18-month-old mice were obtained from preovulatory ovarian follicles. Oocytes were treated with MitoQ and/or BGP-15 during IVM. GV-stage human oocytes were cultured with or without MitoQ. Mitochondrial membrane potential and mitochondrial ROS were measured by live-cell imaging. Meiotic spindle and chromosome alignments were visualized by immunofluorescent labeling of fixed oocytes and the 3-dimensional images were analyzed by Imaris. MAIN RESULTS AND THE ROLE OF CHANCE: MitoQ or BGP-15 during IVM protects against spindle and chromosomal defects in oocytes exposed to oxidative stress and in oocytes from aged mice (P < 0.001). In human oocytes, the presence of MitoQ during IVM promoted nuclear maturation and had a similar positive effect in protecting against chromosomal misalignments (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Our study identifies two excellent candidates that may help to improve fertility in older women. However, these potential therapies must be tested for efficacy in clinical IVM systems, and undergo thorough examination of resultant offspring in preclinical models before utilization. WIDER IMPLICATIONS OF THE FINDINGS: Our results using in-vitro systems for oocyte maturation in both mouse and human provide proof of principle that mitochondrially targeted molecules such as MitoQ and BGP-15 may represent a novel therapeutic approach against maternal aging-related spindle and chromosomal abnormalities. STUDY FUNDING/COMPETING INTEREST(S): The project was financially supported by the National Health and Medical Research Council and Australian Research Council, Australia. U.A.-Z. was supported by the Iraqi Higher Education and Scientific Research Ministry PhD scholarship and O.C. was supported by TUBITAK-1059B191601275. M.P.M. consults for MitoQ Inc. and holds patents in mitochondria-targeted therapies. R.L.R. is an inventor on patents relating to the use of BGP-15 to improve gamete quality. TRIAL REGISTRATION NUMBER: N/A.

Cellular Origins of Endometriosis: Towards Novel Diagnostics and Therapeutics.

Endometriosis remains an enigmatic disease of unknown etiology, with delayed diagnosis and poor therapeutic options. This review will discuss the cellular, physiological, and genomic evidence of Sampson's hypothesis of retrograde menstruation as a cause of pelvic endometriosis and as the basis of phenotypic heterogeneity of the disease. We postulate that collaborative research at the single cell level focused on unlocking the cellular, physiological, and genomic mechanisms of endometriosis will be accompanied by advances in personalized diagnosis and therapies that target unique subtypes of endometriosis disease. These advances will address the clinical conundrums of endometriosis clinical care-including diagnostic delay, suboptimal treatments, disease recurrence, infertility, chronic pelvic pain, and quality of life. There is an urgent need to improve outcomes for women with endometriosis. To achieve this, it is imperative that we understand which cells form the lesions, how they arrive at distant sites, and what factors govern their ability to survive and invade at ectopic locations. This review proposes new research avenues to address these basic questions of endometriosis pathobiology that will lay the foundations for new diagnostic tools and treatment pathways.