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Objective: To assess health outcomes, including growth up to 2 years of age, in children born after embryo vitrification in comparison with children born after fresh embryo transfer. Design: A prospective cohort study. Setting: A single-center university hospital. Patient(s): Singletons born after the transfer of vitrified or fresh embryos, either at the cleavage or blastocyst stage between 2014 and 2018, were included. Intervention(s): Multiple linear and logistic regression analyses were used to study the association between outcomes after vitrified versus fresh embryo transfer, controlling for neonatal, treatment, and maternal characteristics. Subgroup analysis according to cycle protocol (hormone replacement vs. natural cycle) and strategy (freeze-all vs. previous fresh cycle) was also performed. Main Outcome Measure(s): Measurements at birth and growth in infancy and childhood, as well as health outcomes, including congenital malformations, interventions, medication use, and hospitalizations are reported. Result(s): Birth characteristics were available for 1237 and 2063 children born after embryo vitrification and fresh embryo transfer, respectively. Follow-up data were available for 582 and 757 children at infancy and for 233 and 296 children at 2 years, respectively. Birthweight, height, and head circumference SD scores of children born after embryo vitrification were higher than children born after fresh embryo transfer, even after adjustment for neonatal, treatment, and maternal characteristics. In infancy, weight and height SD scores were larger for children born after embryo vitrification, but not after adjustment for covariates. In childhood, no differences in anthropometry were observed between the groups. Weight and height gain from birth to infancy and from infancy to early childhood were comparable between the groups. Comparable rates of severe developmental problems, hospital admissions, surgical interventions, and of chronic medication use were observed up to the age of 2 years. Subgroup analysis showed that growth parameters were not affected by the cycle protocol or strategy at any age. Conclusion(s): Our study indicated that embryo vitrification is associated with higher birthweight, even after controlling for confounders. However, in early childhood, anthropometry and weight and height gain was not different in children born after vitrified or fresh embryo transfer.

The endometrial microbiota composition may be associated with implantation success. However, a ‘core’ composition has not yet been defined. This exploratory study analysed the endometrial microbiota by 16S rRNA sequencing (V1–V2 region) of 141 infertile women whose first IVF/ICSI cycle failed and compared the microbiota profiles of women with and without a live birth within 12 months of follow-up, and by infertility cause and type. Lactobacillus was the most abundant genus in the majority of samples. Women with a live birth compared to those without had significantly higher Lactobacillus crispatus relative abundance (RA) (p = 0.029), and a smaller proportion of them had ≤ 10% L. crispatus RA (42.1% and 70.4%, respectively; p = 0.015). A smaller proportion of women in the male factor infertility group had ≤ 10% L. crispatus RA compared to women in the unexplained and other infertility causes groups combined (p = 0.030). Women with primary infertility compared to secondary infertility had significantly higher L. crispatus RA (p = 0.004); lower proportions of them had ≤ 10% L. crispatus RA (p = 0.009) and > 10% Gardnerella vaginalis RA (p = 0.019). In conclusion, IVF/ICSI success may be associated with L. crispatus RA and secondary infertility with endometrial dysbiosis, more often than primary infertility. These hypotheses should be tested in rigorous well-powered longitudinal studies.

RESEARCH QUESTION: Is there an association between FSHR sequence variants and reproductive outcomes following IVF in predicted normoresponders? DESIGN: Multicentre prospective cohort study conducted from November 2016 to June 2019 in Vietnam, Belgium and Spain including patients aged G, c.2039A>G, c.-29G>A) and one FSHB sequence variants (c.-211G>T). Clinical pregnancy rate (CPR), live birth rate (LBR) and miscarriage rate in the first embryo transfer and cumulative live birth rate (CLBR) were compared between the different genotypes. RESULTS: A total of 351 patients underwent at least one embryo transfer. Genetic model analysis that adjusted for patient age, body mass index, ethnicity, type of embryo transfer, embryo stage and number of top-quality embryos transferred revealed a higher CPR for homozygous patients for the variant allele G of c.919A>G when compared to patients with genotype AA (60.3% versus 46.3%, adjusted odds ratio [ORadj] 1.96, 95% confidence interval [CI] 1.09-3.53). Also, c.919A>G genotypes AG and GG presented a higher CPR and LBR when compared with genotype AA (59.1% versus 46.3%, ORadj 1.80, 95% CI 1.08-3.00, and 51.3% versus 39.0%, ORadj 1.69, 95% CI 1.01-2.80, respectively). Cox regression models revealed a statistically significantly lower CLBR for c.2039A>G genotype GG in the codominant model (hazard ratio [HR] 0.66, 95% CI 0.43-0.99). CONCLUSION: These results demonstrate a previously unreported association between variant c.919A>G genotype GG and higher CPR and LBR in infertile patients and reinforce a potential role for genetic background in predicting the reproductive prognosis following IVF.

Oocyte in vitro maturation (IVM) has been proposed as an alternative to conventional ovarian stimulation (COS) in subfertile women with polycystic ovary syndrome. To evaluate the effectiveness and safety of IVM compared with COS in women with predicted hyperresponse to gonadotropins, we searched the published literature for relevant studies comparing any IVM protocol with any COS protocol followed by in vitro fertilization or intracytoplasmic sperm injection. A systematic review was undertaken on 3 eligible prospective studies. Live birth rate was not significantly lower after IVM vs. COS (odds ratio [95% confidence interval] of 0.56 [0.32-1.01] overall, 0.83 [0.63-1.10] for human chorionic gonadotropin (hCG)-triggered IVM [hCG-IVM] and 0.45 [0.18-1.13] for non-hCG-triggered IVM [non-hCG-IVM]), irrespective of the stage of transferred embryos. Data from nonrandomized studies generally showed either significantly low or statistically comparable rates of live birth with IVM vs. COS. Most studies have not identified any significant difference between IVM and COS with respect to the rates of obstetric or perinatal complications, apart from a potentially higher rate of hypertensive disorders during pregnancy. The development of offspring from IVM and COS with in vitro fertilization or intracytoplasmic sperm injection appears to be similar. Additional research is needed to identify which patient populations will benefit most from IVM, to define the appropriate clinical protocol, and to develop the optimal culture system.

RESEARCH QUESTION : What is the discontinuation rate among patients with remaining cryopreserved embryos in Belgium and what are the reasons for discontinuation? DESIGN : Multicenter, cross-sectional study performed in 11 Belgian fertility clinics (covering 80% of ART population). Patients were eligible (n=1917) in case they underwent an unsuccessful fresh embryo transfer (fresh group) or FET (in between group) and did not start a subsequent FET cycle within one year despite the presence of remaining cryopreserved embryos. Denominator were all patients with embryos cryopreserved during the same period (2012-2017) (n=21329). Data were collected through an online anonymous questionnaire. RESULTS : The discontinuation rate for patients with remaining cryopreserved embryos is 9% (1917/21329). For the final analysis, 304 completed questionnaires were included. The most important checked reasons for discontinuation of FET cycles were psychological (50%) and physical (43%) burden, impact on work (29%), woman's age (25%) and impact on relationship (25%). In 69% of the cases the patient her/himself made the decision to delay FET treatment. In 16% of the respondents, the decision to delay FET was determined by external factors: treating physician (9%), social environment (4%), close family (3%) and society (3%). Suggested improvements by the respondents were psychological support before (41%), during (51%) and after (51%) treatment, as well as lifestyle counseling (44%) and receiving digital information (43%). CONCLUSIONS : The discontinuation rate is remarkably high in patients with remaining cryopreserved embryos, who are considered to have a good prognosis. Respondents stressed the need to better integrate psychological and patient tailored care in our daily ART practice.

STUDY QUESTION Can early pregnancies be accurately and cost-effectively diagnosed and managed using a new medical computerized tool? SUMMARY ANSWER Compared to the standard clinical approach, retrospective implementation of the new medical software in a gynaecological emergency unit was correlated with more accurate diagnosis and more cost-effective management. WHAT IS KNOWN ALREADY Early pregnancy complications are responsible for a large percentage of consultations, mostly in emergency units, with guidelines becoming complex and poorly known/misunderstood by practitioners. STUDY DESIGN, SIZE, DURATION A total of 780 gynaecological emergency consultations (446 patients), recorded between November 2018 and June 2019 in a tertiary university hospital, were retrospectively encoded in a new medical computerized tool. The inclusion criteria were a positive hCG test result, ultrasonographical visualization of gestational sac, and/or embryo corresponding to a gestational age of 14 weeks or less. Diagnosis and management suggested by the new computerized tool are named eDiagnoses, while those provided by a gynaecologist member of the emergency department staff are called medDiagnoses. PARTICIPANTS/MATERIALS, SETTING, METHODS Usability was the primary endpoint, with accuracy and cost reduction, respectively, as secondary and tertiary endpoints. Identical eDiagnoses/medDiagnoses were considered as accurate. During follow-up visits, if the updated eDiagnoses and medDiagnoses became both identical to a previously discrepant eDiagnosis or medDiagnosis, this previous eDiagnosis or medDiagnosis was also considered as correct. Four double-blinded experts reviewed persistent discrepancies, determining the accurate diagnoses. eDiagnoses/medDiagnoses accuracies were compared using McNemar’s Chi square test, sensitivity, specificity, and predictive values. MAIN RESULTS AND THE ROLE OF CHANCE Only 1 (0.1%) from 780 registered medical records lacked data for full encoding. Out of the 779 remaining consultations, 675 eDiagnoses were identical to the medDiagnoses (86.6%) and 104 were discrepant (13.4%). From these 104, 60 reached an agreement during follow-up consultations, with 59 medDiagnoses ultimately changing into the initial eDiagnoses (98%) and only one discrepant eDiagnosis turning later into the initial medDiagnosis (2%). Finally, 24 remained discrepant at all subsequent checks and 20 were not re-evaluated. Out of these 44, the majority of experts agreed on 38 eDiagnoses (86%) and 5 medDiagnoses (11%, including four twin pregnancies whose twinness was the only discrepancy). No majority was reached for one discrepant eDiagnosis/medDiagnosis (2%). In total, the accuracy of eDiagnoses was 99.1% (675 + 59 + 38 = 772 eDiagnoses out of 779), versus 87.4% (675 + 1 + 5 = 681) for medDiagnoses (P LIMITATIONS, REASONS FOR CAUTION The retrospective study design is a limitation. Some observed improvements with the medical software could derive from the encoding by a rested and/or more experienced physician who had a better ultrasound interpretation. This software cannot replace clinical and ultrasonographical skills but may improve the compliance to published guidelines. WIDER IMPLICATIONS OF THE FINDINGS This medical computerized tool is improving. A new version considers diagnosis and management of multiple pregnancies with their specificities (potentially multiple locations, chorioamnionicity). Prospective evaluations will be required. Further developmental steps are planned, including software incorporation into ultrasound devices and integration of previously published predictive/prognostic factors (e.g. serum progesterone, corpus luteum scoring). STUDY FUNDING/COMPETING INTEREST(S) No external funding was obtained for this study. F.B. and D.G. created the new medical software. TRIAL REGISTRATION NUMBER NCT03993015

This study aimed to investigate whether female BRCA1- and BRCA2 mutation carriers have a reduced ovarian reserve status, based on serum anti-Mullerian hormone (AMH) levels, antral follicle count (AFC) and ovarian response to ovarian hyperstimulation. A prospective, multinational cohort study was performed between October 2014 and December 2019. Normo-ovulatory women, aged 18–41 years old, applying for their first PGT-cycle for reason of a BRCA mutation (cases) or other genetic diseases unrelated to ovarian reserve (controls), were asked to participate. All participants underwent a ICSI-PGT cycle with a long-agonist protocol for controlled ovarian hyperstimulation. Linear and logistic regression models were used to compare AMH, AFC and ovarian response in cases and controls. Sensitivity analyses were conducted on BRCA1- and BRCA2 mutation carrier subgroups. Thirty-six BRCA mutation carriers (18 BRCA1- and 18 BRCA2 mutation carriers) and 126 controls, with mean female age 30.4 years, were included in the primary analysis. Unadjusted median AMH serum levels (IQR) were 2.40 (1.80–3.00) ng/ml in BRCA mutation carriers and 2.15 (1.30–3.40) ng/ml in controls (p = 0.45), median AFC (IQR) was 15.0 (10.8–20.3) and 14.5 (9.0–20.0), p = 0.54, respectively. Low response rate was 22.6% among BRCA mutation carriers and 9.3% among controls, p = 0.06. Median number of retrieved oocytes was 9 (6–14) in carriers and 10 (7–13) in controls, p = 0.36. No substantial differences were observed between BRCA1- and BRCA2 mutation carriers. Based on several biomarkers, no meaningful differences in ovarian reserve status were observed in female BRCA mutation carriers compared to controls in the context of ICSI-PGT treatment.

OBJECTIVE: Does the dose or type of gonadotropin affect the reproductive outcomes of poor responders undergoing IVF in a modified natural cycle (MNC-IVF)? STUDY DESIGN: This is a retrospective cohort study including patients attending a tertiary referral University Hospital from 1st January 2017 until 1st March 2020. All predicted poor responders (Poseidon groups 3 and 4) who underwent MNC-IVF in our center were included. Mild ovarian stimulation (rFSH/uFSH/hp-hMG) was started when a follicle with a mean diameter of 12-14 mm was observed on ultrasound scan; GnRH antagonist was added from the next day onwards. Mature oocytes were inseminated using ICSI. RESULTS: In total 484 patients undergoing 1398 cycles were included. Mean (SD) age and serum AMH were 38.2 (3.7) years and 0.28 (0.26) ng/ml, respectively. The daily dose of gonadotropins was either

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

RESEARCH QUESTION: Does additional supplementation with oral dydrogesterone improve reproductive outcomes in patients with low serum progesterone concentrations on the day of frozen embryo transfer (FET) after artificial (HRT) endometrial preparation? DESIGN: Retrospective, single-centre cohort study including 694 unique patients performing single blastocyst transfer in an HRT cycle. For luteal phase support, intravaginal micronized vaginal progesterone (MVP, 400 mg twice daily) was administered. Serum progesterone concentrations were assessed prior to FET and outco-mes were compared among patients with normal serum progesterone (≥8.8 ng/ml) continuing the routine protocol and patients with low serum progesterone (

PURPOSE OF REVIEW: The technical improvements in IVF allowed the implementation of nonconventional ovarian stimulation protocols for some specific patients. Where time is crucial, such as with oncologic patients, poor-prognosis patients, patients with low ovarian reserve, and those with advanced maternal age, access to IVF treatment is even more critical. Some of these protocols might start in the late follicular phase, luteal phase, or involve both stimulations within the same ovarian cycle. RECENT FINDINGS: Until now, published evidence showed that oocytes retrieved from unconventional protocol seem to be developmentally, genetically, and reproductively competent. Second stimulation in the same ovarian cycle after the conventional approach may represent a sound alternative to oocyte accumulation. This can be proposed in progress after careful counselling focused on the patients' chances of finding at least one euploid embryo on account of their age and of the number of blastocysts obtained after the conventional approach. SUMMARY: The adoption of these new strategies, known as double stimulation protocol, can be conceived as a real full-personalization of ovarian stimulation. Multicentre prospective RCTs are urgently needed to evaluate the efficacy, efficiency, and costs of double stimulation versus two consecutive conventional approaches with standard or mild stimulation and in a different IVF setting.

Background and Aim In view of their frequent onset during childbearing years, the impact of inflammatory bowel diseases [IBD] on reproductive health is of important concern to young women and to the IBD physician. This study aims to assess the fertility and assisted reproductive technologies outcomes in non-surgically treated IBD females. Methods A systematic review was conducted using MEDLINE, SCOPUS, and EMBASE [until March 2022] to identify studies assessing fertility and assisted reproductive technologies outcomes in women with non-operated IBD, compared with non-IBD patients. Two reviewers independently selected studies, assessed risk of bias, and extracted study data. Results A total of 14 studies encompassing 18 012 patients with ulcerative colitis [UC] and 14 353 patients with Crohn’s disease [CD] were included for analysis. The fertility rate in UC patients and in the general population was comparable, but UC patients tended to have fewer children, mainly by choice. On the contrary, the fertility of CD patients appeared to be reduced. Although a deliberate component cannot be not excluded, the disease itself could affect fertility. Disease activity was associated with reduced fertility in both UC and CD patients. In CD, the colonic involvement of the disease and perianal damage could be associated with subfertility, but data are less consistent. According to the only study reporting the assisted reproductive technologies outcomes, pregnancy rates after in vitro fertilization in subfertile non-operated UC patients and non-IBD patients were similar. Conclusions There is low-quality evidence from observational studies that patients with CD and relapsing UC may have impaired fertility. After assisted reproductive technologies, pregnancy rates of subfertile non-operated UC patients were similar to those of the general population, although this observation requires further scrutiny in larger studies that should include UC and CD patients.

It is generally accepted that microorganisms can colonize a non-pathological endometrium. However, in a clinical setting, endometrial samples are always collected by passing through the vaginal–cervical route. As such, the vaginal and cervical microbiomes can easily cross-contaminate endometrial samples, resulting in a biased representation of the endometrial microbiome. This makes it difficult to demonstrate that the endometrial microbiome is not merely a reflection of contamination originating from sampling. Therefore, we investigated to what extent the endometrial microbiome corresponds to that of the vagina, applying culturomics on paired vaginal and endometrial samples. Culturomics could give novel insights into the microbiome of the female genital tract, as it overcomes sequencing-related bias. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy were included. An additional vaginal swab was taken from each participant right before hysteroscopy. Both endometrial biopsies and vaginal swabs were analyzed using our previously described WASPLab-assisted culturomics protocol. In total, 101 bacterial and two fungal species were identified among these 10 patients. Fifty-six species were found in endometrial biopsies and 90 were found in vaginal swabs. On average, 28 % of species were found in both the endometrial biopsy and vaginal swab of a given patient. Of the 56 species found in the endometrial biopsies, 13 were not found in the vaginal swabs. Of the 90 species found in vaginal swabs, 47 were not found in the endometrium. Our culturomics-based approach sheds a different light on the current understanding of the endometrial microbiome. The data suggest the potential existence of a unique endometrial microbiome that is not merely a presentation of cross-contamination derived from sampling. However, we cannot exclude cross-contamination completely. In addition, we observe that the microbiome of the vagina is richer in species than that of the endometrium, which contradicts the current sequence-based literature.

BackgroundWith the refinement of cryopreservation technology, the number of frozen-warmed embryo transfer (FET) cycles and cryopreserved embryos has increased rapidly. However, studies investigating the effect of storage duration on pregnancy outcomes after vitrification are limited and their results are controversial. Furthermore, the available studies did not take patients’ demographic nor clinical treatment characteristics into account and the cryo-storage duration was short. So this study aimed to explore the effect of storage duration of vitrified warmed embryos on pregnancy and neonatal outcomes in patients with good prognosis and long storage duration of vitrified embryos.MethodsThis study was a bi-centre, retrospective study including 1037 women undergoing their first FET cycles following a fresh cycle from January 2012 until December 2021. Patients were divided into four storage groups in accordance with the storage duration of transferred embryos (612 patients in group I, with storage duration between 1 and 6 months; 202 patients in group II, with storage duration between 7 and 12 months; 141 patients in group III, with storage duration between 13 and 36 months; and 76 patients in group IV, with storage duration between 37 and 84 months). The pregnancy and neonatal outcomes were compared amongst different storage duration groups.ResultsAmongst the different groups, no significant differences were observed in the pregnancy outcomes, including biochemical pregnancy rate, implantation rate, clinical pregnancy rate, ongoing pregnancy rate and live birth rate. In addition, no evidence of differences amongst different storage duration groups was observed in terms of preterm birth, birth length and low birthweight.ConclusionsThe pregnancy and neonatal outcomes of embryos after vitrification were not impaired by storage duration up to 7 years.

For more than two decades, the European IVF-Monitoring Consortium has collected data on IVF in Europe with the aim of monitoring the quality and safety of assisted reproductive technology (ART) treatments, to ensure the highest performance with the lowest risk for patients and their offspring. Likewise, the Society for Assisted Reproductive Technology in the USA and the Australia/New Zealand Assisted Reproduction Database collect, process and publish data in their regions. The better the legal framework for ART surveillance, the more complete and reliable are the datasets. Worldwide, the landscape of ART regulation is fragmented, and until there is a legal obligation to report ART data in all countries, with an appropriate quality control of the data collected, the reported outcomes should be interpreted with caution. Once uniform and harmonized data are achieved, consensus reports based on collective findings can begin to address key topics such as cycle segmentation and complications. Improved registration systems and datasets allowing optimized surveillance should be developed in collaboration with patient representatives to consider patients' needs, especially aiming to provide higher transparency around ART services. Support from national and international reproductive medicine societies will also be essential to the future evolution of ART registries.

Research question: Is intratesticular xenotransplantation a potential ex-vivo model for studying testicular fibrosis related to Klinefelter syndrome? Study design: First, a feasibility study of an ex-vivo model to study testicular fibrosis in patients with Klinefelter syndrome was performed. Testis tissue from boys with pre-pubertal Klinefelter syndrome (n = 3) and controls (n = 2) (

STUDY QUESTION Does addition of choriogonadotropin beta (recombinant CG beta) to follitropin delta increase the number of good-quality blastocysts following ovarian stimulation in a long GnRH agonist protocol? SUMMARY ANSWER At the doses investigated, the addition of CG beta reduced the number of intermediate follicles and related down-stream parameters including the number of oocytes and blastocysts. WHAT IS KNOWN ALREADY CG beta is a novel recombinant hCG (rhCG) molecule expressed by a human cell line (PER.C6®) and has a different glycosylation profile compared to urinary hCG or rhCG derived from a Chinese Hamster Ovary (CHO) cell line. In the first-in-human trial, the CG beta pharmacokinetics were similar between men and women. In women, the AUC and the peak serum concentration (Cmax) increased approximately dose proportionally following single and multiple daily doses. In men, a single dose of CG beta provided higher exposure with a longer half-life and proportionately higher testosterone production than CHO cell-derived rhCG. STUDY DESIGN, SIZE, DURATION This placebo-controlled, double-blind, randomized trial (RAINBOW) was conducted in five European countries to explore the efficacy and safety of CG beta as add-on treatment to follitropin delta in women undergoing ovarian stimulation in a long GnRH agonist protocol. Randomization was stratified by centre and age (30–37 and 38–42 years). The primary endpoint was the number of good-quality blastocysts (Grade 3 BB or higher). Subjects were randomized to receive either placebo or 1, 2, 4, 8 or 12 µg CG beta added to the daily individualized follitropin delta dose during ovarian stimulation. PARTICIPANTS/MATERIALS, SETTING, METHODS In total, 620 women (30–42 years) with anti-Müllerian hormone (AMH) levels between 5 and 35 pmol/l were randomized in equal proportions to the six treatment groups and 619 subjects started treatment. All 619 subjects were treated with an individualized dose of follitropin delta determined based on AMH (Elecsys AMH Plus Immunoassay) and body weight. Triggering with rhCG was performed when 3 follicles were ≥17 mm but no more than 25 follicles ≥12 mm were reached. MAIN RESULTS AND THE ROLE OF CHANCE The demographic characteristics were comparable between the six treatment groups and the overall mean age, body weight and AMH were 35.6 ± 3.3 years, 65.3 ± 10.7 kg and 15.3 ± 7.0 pmol/l, respectively. The incidence of cycle cancellation (range 0–2.9%), total follitropin delta dose (mean 112 µg) and duration of stimulation (mean 10 days) were similar across the groups. At stimulation Day 6, the number and size of follicles was similar between the treatment groups, whereas at the end-of-stimulation dose-related decrease of the intermediate follicles between 12 and 17 mm was observed in comparison to the placebo group. In contrast, the number of follicles ≥17 mm was similar between the CG beta dose groups and the placebo group. A reduced number of intermediate follicles (12 to 17 mm) and fewer oocytes (mean range 9.7 to 11.2) were observed for all doses of CG beta compared to the follitropin delta only group (mean 12.5). The mean number of good-quality blastocysts was 3.3 in the follitropin delta group and ranged between 2.1 and 3.0 across the CG beta groups. The incidence of transfer cancellation was higher in the 4, 8 and 12 µg group, mostly as no blastocyst was available for transfer. In the group receiving only follitropin delta, the ongoing pregnancy rate (10–11 weeks after transfer) was 43% per started cycle versus 28–39% in CG beta groups and 49% per transfer versus 38–50% in the CG beta groups. There was no apparent effect of CG beta on the incidence of adverse events, which was 48.1% in the placebo group and 39.6–52.3% in the CG beta dose groups. In line with the number of collected oocytes, the overall ovarian hyperstimulation syndrome incidence remained lower following follitropin delta with CG beta (2.0–10.3%) compared with follitropin delta only treatment (11.5%). Regardless of the dose, CG beta was safe and well-tolerated with low risk of immunogenicity. LIMITATIONS, REASONS FOR CAUTION The effect of the unique glycosylation of CG beta and its associated potency implications in women were not known prior to this trial. Further studies will be needed to evaluate optimal doses of CG beta for this and/or different indications. WIDER IMPLICATIONS OF THE FINDINGS The high ongoing pregnancy rate in the follitropin delta group supports the use of individualized follitropin delta dosing in a long GnRH agonist protocol. The addition of CG beta reduced the presence of intermediate follicles with the investigated doses and negatively affected all down-stream parameters. Further clinical research will be needed to assess the optimal dose of CG beta in the optimal ratio to follitropin delta to develop this novel combination product containing both FSH and LH activity for ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by Ferring Pharmaceuticals, Copenhagen, Denmark. B.M. and P.L. are employees of Ferring Pharmaceuticals. M.F.S., H.V., C.Y.A., M.F., C.B., A.P. and Y.K. have received institutional clinical trial fees from Ferring Pharmaceuticals. C.B. has received payments for lectures from Organon, Ferring Pharmaceuticals, Merck A/S and Abbott. M.F.S. has received payment for lectures from Ferring Pharmaceuticals. Y.K. has received payment for lectures from Merck and travel support from Gedeon Richter. H.V. has received consulting fees from Oxo and Obseva and travel support from Gedeon Richter, Ferring Pharmaceuticals and Merck. C.Y.A. has received payment for lectures from IBSA, Switzerland. M.F and C.Y.A. were reimbursed as members of the Data Monitoring Board in this trial. M.F. has an issued patent about unitary combination of FSH and hCG (EP1633389). TRIAL REGISTRATION NUMBER 2017-003810-13 (EudraCT Number) TRIAL REGISTRATION DATE 21 May 2018 DATE OF FIRST PATIENT’S ENROLMENT 13 June 2018

Objectives To study the presence of viral RNA in follicular fluid, cumulus cells and endometrial tissue samples in SARS-CoV-2 positive women undergoing assisted reproductive technologies (ART). Design Prospective, single-center, observational study. Setting Tertiary hospital, University Hospital UZ Brussel, Belgium. Patients Sixteen patients undergoing transvaginal oocyte retrieval who had a positive SARS-CoV-2 RNA test less than 48 hours before the procedure. All cases were done between September 2020 and June 2021 and used in-vitro fertilization or intracytoplasmic sperm injection. All embryos were vitrified to avoid conception during SARS-CoV-2 infection. Intervention Follicular fluid aspirated during oocyte retrieval, cumulus cells and endometrial samples were analyzed for SARS-CoV-2 RNA using RealStar® SARS-CoV-2 RT-PCR-Kit1.0 (Altona-Diagnostics). Main outcome measures The primary outcome parameter was the detection of viral RNA in follicular fluid, cumulus cells and endometrial cells. Fertilization rate, embryo developmental potential and clinical outcome after frozen embryo transfer were secondary outcome parameters. Results Samples from sixteen patients were analyzed. Cycle threshold (Ct) values < 40 were considered positive. All samples were negative for SARS-CoV-2 viral RNA. No inflammatory lesions of the endometrium were identified histologically. Fertilization rate, embryo development and clinical outcomes after embryo transfer were reassuring. Conclusions In women infected with SARS-CoV-2 who underwent ART, viral RNA was undetectable in follicular fluid, cumulus cells and endometrium. Caution is warranted in view of the small sample size, and the risk of SARS-CoV-2 affecting the embryo via ART cannot be ruled out. Adequate counselling of women and couples undergoing ART is crucial in parallel with further research on the effect of exposure of the early human embryo to SARS-CoV-2., In patients with a SARS-CoV-2 infection, viral RNA was undetectable in follicular fluid, cumulus cells and endometrial biopsies. Additionally, embryological data and clinical outcomes are reported.

Humans present remarkable diversity in their mitochondrial DNA (mtDNA) in terms of variants across individuals as well as across tissues and even cells within one person. We have investigated the timing of the first appearance of this variant-driven mosaicism. For this, we deep-sequenced the mtDNA of 254 oocytes from 85 donors, 158 single blastomeres of 25 day-3 embryos, 17 inner cell mass and trophectoderm samples of 7 day-5 blastocysts, 142 bulk DNA and 68 single cells of different adult tissues. We found that day-3 embryos present blastomeres that carry variants only detected in that cell, showing that mtDNA mosaicism arises very early in human development. We classified the mtDNA variants based on their recurrence or uniqueness across different samples. Recurring variants had higher heteroplasmic loads and more frequently resulted in synonymous changes or were located in non-coding regions than variants unique to one oocyte or single embryonic cell. These differences were maintained through development, suggesting that the mtDNA mosaicism arising in the embryo is maintained into adulthood. We observed a decline in potentially pathogenic variants between day 3 and day 5 of development, suggesting early selection. We propose a model in which closely clustered mitochondria carrying specific mtDNA variants in the ooplasm are asymmetrically distributed throughout the cell divisions of the preimplantation embryo, resulting in the earliest form of mtDNA mosaicism in human development.

Research question Do cumulative live birth rates (CLBR) differ between polycystic ovary syndrome (PCOS) phenotypes when a freeze-all strategy is used to prevent ovarian hyperstimulation syndrome (OHSS) after ovarian stimulation (OS)? Design This is a single-centre, retrospective cohort study including 422 women with PCOS as defined by Rotterdam criteria or with PCO-like ovarian morphology-only (PCOM) in whom, between January 2015 and December 2019, a freeze-all strategy was applied after GnRH agonist triggering in the context of hyper-response defined as ≥18 follicles of ≥11 mm in their first or second IVF/ICSI cycle in a tertiary referral hospital. The primary outcome was CLBR resulting from the transfer of all cryopreserved embryos from the same IVF/ICSI cycle. Analysis was performed by multivariate logistic regression analysis adjusting for confounders. Results In total, 91/422 (21.6%) patients had PCOS phenotype A (hyperandrogenism + ovulation disorder + PCOM = HOP), 33/422 (7.8%) had phenotype C (hyperandrogenism + PCOM = HP), 161/422 (38.2%) had phenotype D (ovulation disorder + PCOM = OP) and 137/422 (32.5%) had PCOM. Unadjusted CLBR was similar among the groups (69.2%, 69.7%, 79.5% and 67.9%, respectively; p= 0.11). The multivariate logistic regression model adjusting for age, BMI, number of oocytes, fertilization rate and embryo stage at vitrification confirmed that the PCOS/PCOM phenotype did not have any impact on CLBR [OR 0.72, CI 0.24-2.14 (phenotype C); OR 1.55, CI 0.71-3.36 (phenotype D); OR 0.84, CI 0.39-1.83 (PCOM); p= 0.2, with phenotype A as reference]. Conclusions In subfertile women with PCOS eligible for ART, hyper-response after OS confers excellent cumulative live birth rates of ∼70%, irrespective of the phenotype, when a freeze-all strategy is used. This contrasts with the unfavourable clinical outcomes that have previously been observed in hyperandrogenic PCOS women who underwent mild OS targeting normal ovarian response and fresh embryo transfer. The data should however be interpreted with caution as the retrospective nature of the study holds the possibility of unmeasured confounding factors. The results cannot be generalized to all ART cycles in women with polycystic ovaries as they pertain to those cycles in which hyper-response is observed after OS.

Purpose: To report the case of a young woman diagnosed with Turner syndrome (TS) who achieved a live birth using her own oocytes that had been vitrified for fertility preservation. Methods: A 25-year-old woman with mosaic (45,X/46,XX) TS was referred for fertility preservation (FP) counseling. Serum anti-Müllerian hormone (AMH) level was normal (6.4 µg/L). In view of the unpredictable rate of follicle loss in TS individuals, she requested FP and underwent two cycles of ovarian stimulation (OS) for oocyte cryopreservation (OoC) using a GnRH antagonist protocol and recombinant follicle stimulating hormone (rFSH), 200-250 IU daily for 8 resp. 12 days. Results: In total, 29 metaphase II oocytes (MII) were vitrified after OS. After conceiving spontaneously and achieving a live birth, she returned to the clinic five years after OoC with a desire for pregnancy using in vitro fertilization (IVF) of her cryopreserved oocytes and preimplantation genetic testing (PGT-A). All 29 MII oocytes were thawed; 23 oocytes survived (79.3%) and were inseminated with partner sperm using intracytoplasmic sperm injection (ICSI). Thirteen oocytes were fertilized resulting in three good quality blastocysts which were vitrified after trophectoderm biopsy for PGT-A using array-CGH. Two blastocysts were found to be euploid. One was thawed and transferred to the uterus using a HRT priming protocol. An uneventful pregnancy occurred. The patient delivered a healthy baby girl weighing 3490 g at 40 weeks of gestation. Conclusions: We report the first live birth achieved using cryopreserved oocytes in a woman diagnosed with mosaic TS. Cryopreservation of oocytes after ovarian stimulation is a realistic option for FP in selected post menarche individuals with mosaic TS. Whether PGT-A may reduce the risk of pregnancy loss in TS has to be confirmed by further studies.

About half of testicular sperm extraction (TESE) procedures in men with non-obstructive azoospermia (NOA), including men with Klinefelter syndrome (KS), are unsuccessful. To avoid unnecessary invasive surgery, biomarkers for spermatozoa were studied. In addition, markers for spermatogonia in testis tissue were explored. This study aimed to find biomarkers in the semen and/or urine of NOA patients to predict the presence of spermatogonia in the testis. Differentially expressed miRNAs were identified (1) between samples from patients with and without a positive TESE procedure as well as (2) between TESE-negative patients with and without spermatogonia. A total of thirteen upregulated miRNAs (ten in seminal plasma and three in urine) were found in the TESE-negative/spermatogonia-positive group compared to the TESE-negative/spermatogonia-negative group. These miRNAs could be potential biomarkers for spermatogonia; however, more research is necessary to validate their predictive power.

A high ovarian response to conventional ovarian stimulation (OS) is characterized by an increased number of follicles and/or oocytes compared with a normal response (10-15 oocytes retrieved). According to current definitions, a high response can be diagnosed before oocyte pick-up when >18-20 follicles ≥11-12 mm are observed on the day of ovulation triggering; high response can be diagnosed after oocyte pick-up when >18-20 oocytes have been retrieved. Women with a high response are also at high risk of early ovarian hyper-stimulation syndrome (OHSS)/or late OHSS after fresh embryo transfers. Women at risk of high response can be diagnosed before stimulation based on several indices, including ovarian reserve markers (anti-Müllerian hormone [AMH] and antral follicle count [AFC], with cutoff values indicative of a high response in patients with PCOS of >3.4 ng/mL for AMH and >24 for AFC). Owing to the high proportion of high responders who are at the risk of developing OHSS (up to 30%), this educational article provides a framework for the identification and management of patients who fall into this category. The risk of high response can be greatly reduced through appropriate management, such as individualized choice of the gonadotropin starting dose, dose adjustment based on hormonal and ultrasound monitoring during OS, the choice of down-regulation protocol and ovulation trigger, and the choice between fresh or elective frozen embryo transfer. Appropriate management strategies still need to be defined for women who are predicted to have a high response and those who have an unexpected high response after starting treatment. ispartof: BEST PRACTICE & RESEARCH CLINICAL OBSTETRICS & GYNAECOLOGY vol:86 ispartof: location:Netherlands status: published

Although a wealth of data has been published regarding fertility preservation (FP) in women with malignant diseases who receive gonadotoxic treatment, the role of FP in non-malignant conditions has been studied to a much lesser extent. These include benign haematological, autoimmune, and genetic disorders, as well as a multitude of benign gynaecological conditions (BGCs) that may compromise ovarian reserve and/or reproductive potential due to pathogenic mechanisms or as a result of medical or surgical treatments. Alongside accumulating data that document the reproductive potential of cryopreserved oocytes and ovarian tissue, there is potential interest in FP for women with BGCs at risk of infertility; however, there are currently insufficient data about FP in women with BGCs to develop guidelines for clinical practice. The purpose of this article is to appraise the available evidence regarding FP for BGC and discuss potential strategies for FP based on estimated ovarian impairment and on short-term and long-term reproductive goals of patients. Cost-effectiveness considerations and patients’ perspectives will also be discussed.

BACKGROUND AND AIMS: Inflammatory bowel disease (IBD) can affect patients during their childbearing years. Literature evidence is scarce regarding the level of knowledge among health care professionals (HCPs) and patients about the impact of IBD on fertility. The aim of this survey was to investigate HCPs' and patients' knowledge on fertility, pregnancy, and sexual function, to evaluate how HCPs approach this topic and to report patients' reproductive outcomes. METHODS: Subjects were invited to anonymously complete an online questionnaire collecting data on demographics, patients' disease characteristics, Crohn's and colitis pregnancy-specific disease-related knowledge (CCPKnow), family planning, reason of childlessness, pregnancy outcomes, need for assisted reproductive technology, impact on sexual function, and availability of patients' information regarding IBD and pregnancy. RESULTS: A total of 257 HCPs from 40 countries and 793 patients (615 females, 176 males and 2 who preferred not to disclose their gender; 396 (50%) with ulcerative colitis, 381 (48%) with Crohn's disease, 14 (1.8%) with undetermined IBD) from 4 countries completed the survey. In total, 98.4% of HCPs had good or very good pregnancy-specific knowledge according to CCPKnow score, compared to only 29.3% of patients. Of the women surveyed, 56.3% had no children (14.1% due to a voluntary choice). A total of 427 pregnancies and 401 live births were reported in 266 women. Twenty-four pregnancies (5.6%) in 22 women required assisted reproductive technologies (ART). There were no more complications in pregnancies resulting from ART compared with spontaneous conception (5/24; 20.8% vs 81/401; 20.2%). Three quarters of IBD patients (75.6%) had breastfed. An impaired sexual function was found in one-fifth (21.9%) of men with IBD, while two-thirds (66.1%) of the women reported sexual function impairment. Surprisingly, 63% of patients reported not having received any information about IBD and pregnancy, and only 10% of patients had received information from their IBD specialist. In addition, 42.1% and 36% of HCPs had already referred a patients to a medically assisted reproduction center to receive general information about their reproductive health and about options of fertility preservation (e.g., cryopreservation), respectively. CONCLUSION: IBD patients have a poor knowledge about the impact of IBD on fertility and pregnancy and HCPs do not sufficiently inform their patients. More information on these topics is needed for IBD patients.

Chronic endometritis is a poorly understood infectious or inflammatory process, potentially disrupting the correct implantation of a human embryo (Puente et al., 2020). The exact prevalence is a subject of discussion and ranges across the available literature from 2% to almost 60%, with a higher suspicion of the condition being present in women with recurrent early pregnancy loss and recurrent implantation failure (Puente et al., 2020). The impact of chronic endometritis on reproductive outcomes following IVF remains questionable given the lack of proper data convincingly showing an improvement after diagnosis and treatment. This article aims to provide the reader with a critical appraisal of current diagnostic methods, treatments and patient populations to be tested for chronic endometritis.

ObjectiveTo observe the real-world utilization patterns, effectiveness and safety profile of follitropin delta in women ≥18 years naïve to ovarian stimulation undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).DesignProspective, multinational, multicenter, observational study. All IVF/ICSI treatment protocols were conducted according to routine clinical practice, including undertaking fresh/frozen transfers. Outcomes included use of dosing algorithm, follitropin delta dosing patterns, ovarian response, pregnancy rates and adverse drug reactions (ADRs).ResultsThe first ovarian stimulation cycle using follitropin delta was initiated in 944 women. Mean baseline demographics were: age, 33.5 ± 4.7 years; bodyweight, 67.1 ± 13.6 kg; anti-Müllerian hormone, 20.3 ± 16.1 pmol/L (2.84 ± 2.25 ng/mL). The dosing algorithm was used to calculate the follitropin delta daily starting dose in 893/944 women (94.5%). The mean difference between the calculated and prescribed daily dose was small (0.2 ± 1.40 µg). The mean daily starting follitropin delta dose was 10.4 ± 2.72 µg and the mean total dose administered was 104 µg. Follitropin delta dose adjustments were reported for 57/944 (6.0%) women. The mean number of retrieved oocytes was 10.1 ± 7.03. Ongoing pregnancy at 10–11 weeks was reported for 255 women (27.0% per initiated cycle and 43.1% per fresh transfer [n=592]). Cumulative ongoing pregnancy rate after fresh and/or frozen transfer was 36.4% (344/944). Four women discontinued follitropin delta due to ADRs. Ovarian hyperstimulation syndrome (OHSS) was the most frequently reported ADR (n=37 [3.9%]); most cases of OHSS were of mild or moderate intensity (n=30 [3.2%]).ConclusionsThis large real-world study of follitropin delta utilization patterns confirms its good pregnancy rates while minimizing OHSS risk during first ovarian stimulation cycle.

Background: Klinefelter syndrome (KS; 47,XXY) affects 1-2 in 1000 males. Most KS men suffer from an early germ cell loss and testicular fibrosis from puberty onwards. Mechanisms responsible for these processes remain unknown. Previous genomics studies on KS tissue focused on germ cell loss, while a transcriptomic analysis focused on testicular fibrosis has not yet been performed. This study aimed to identify factors involved in the fibrotic remodelling of KS testes by analysing the transcriptome of (non-)fibrotic testicular tissue.Material and methods: RNA sequencing was performed to compare the genetic profile of testicular samples with (KS and testis atrophy) and without (Sertoli cell-only syndrome and fertile controls) fibrosis (n=5, each). Additionally, differentially expressed genes (DEGs) between KS and testis atrophy samples were studied to reveal KS-specific fibrotic genes. DEGs were considered significant when p2. Next, downstream analyses (GO and KEGG) were performed. Lastly, RNA scope was performed to validate the results.Results: The first analysis (fibrotic vs non-fibrotic) resulted in 734 significant DEGs (167 up- and 567 down-regulated). Genes involved in the extracellular structure organization (e.g. VCAM1) were found upregulated. KEGG analysis showed an up-regulation of genes involved in the TGF-β pathway.The KS vs TA analysis resulted in 539 significant DEGs (59 up- and 480 down-regulated). Chronic inflammatory response genes were found upregulated. The overlap of X-linked DEGs from the two analyses revealed three genes: matrix-remodelling associated 5 (MXRA5), doublecortin (DCX) and variable charge X-Linked 3B (VCX3B). RNA in situ hybridization showed an overexpression of VCAM1, MXRA5 and DCX within the fibrotic group compared with the non-fibrotic group.Conclusion: In this study, DEGs between fibrotic and non-fibrotic tissue were first compared, revealing fibrotic genes, including VCAM1. Secondly, DEGs between KS and TA samples were studied and the analysis revealed KS-specific, fibrotic genes. When comparing X-linked genes of the first analysis (fibrotic versus non-fibrotic) with those of the second analysis (KS versus TA), KS-related X-linked fibrotic genes were revealed, i.e. MXRA5, DCX and VCX3B. Their potential role in KS-related testicular fibrosis needs further study.

Objective Urine self-sampling has gained increasing interest for cervical cancer screening. In contrast to analytical performance, little information is available regarding the clinical accuracy for high-risk Human Papillomavirus (hrHPV) testing on urine. Methods VALHUDES is a diagnostic test accuracy study comparing clinical accuracy to detect high-grade cervical precancer (CIN2+) of HPV testing on self-collected compared to clinician-collected samples ( NCT03064087 ). Disease outcome was assessed by colposcopy and histology. The Abbott RealTime High Risk HPV assay performance was evaluated on Colli-Pee collected first-void urine with cervical outcomes as comparator. Results As no assay cut-off for urine has been clinically validated, we used the predefined cut-off for cervical samples (CN ≤ 32). Using this cut-off, hrHPV testing was similarly sensitive (relative sensitivity 0.95; 95% CI: 0.88–1.01) and specific (relative specificity 1.03; 95% CI: 0.95–1.13) for detection of CIN2+ compared to testing cervical samples. In the subgroup of women of 30 years and older, similar relative sensitivity (0.97; 95% CI: 0.89–1.05) and specificity (1.02; 95% CI: 0.93–1.12) was found. Additionally, an exploratory cut-off (CN ≤ 33.86) was defined which further improved sensitivity and analytical test performance. Conclusion HrHPV-DNA based PCR testing on home-collected first-void urine has similar accuracy for detecting CIN2+ compared to cervical samples taken by a clinician.

Research question : What are the most pressing educational needs of fertility healthcare professionals using assisted reproductive technologies (ART)? Design : This mixed-methods study combined qualitative interviews with quantitative surveys. Participants included physicians and nurses specialised in reproductive endocrinology or in obstetrics/gynaecology, and laboratory specialists, with a minimum of three years of experience, practicing in Australia, Brazil, Canada, China, France, Germany, India, Italy, Japan, Mexico, Spain, or the UK. Maximum variation purposive sampling was used to ensure a mix of experience and settings. Interviews were transcribed and coded through thematic analysis. Quantitative data were analysed using frequency tables, cross-tabulations and Chi-square tests to compare results by reimbursement context. Results : A total of 535 participants were included (273 physicians, 145 nurses and 117 laboratory specialists). Knowledge gaps, skill gaps, and attitude issues were identified in relation to: 1) ovarian stimulation (e.g. knowledge of treatments and instruction protocols for ovarian stimulation), 2) embryo culture and cryopreservation / vitrification (e.g., diverging opinions on embryo freezing, 3) embryo assessment (e.g., performing genetic testing), 4) support of luteal phase and optimising pregnancy outcomes (e.g., knowledge of assessment methods for endometrial receptivity), and 5) communication with patients (e.g., reluctance to address emotional distress). Conclusions : This descriptive, exploratory study corroborates previously reported gaps in fertility care and identify potential causes of these gaps. Findings provide evidence to inform educational programmes for healthcare professionals who use ART in their practice and calls for the development of case-based education and interprofessional training program to improve care for patients with fertility issues.

STUDY QUESTION Does the presence of FSHR single-nucleotide polymorphisms (SNPs) affect late follicular phase progesterone and estradiol serum levels in predicted normoresponders treated with rFSH? SUMMARY ANSWER The presence of FSHR SNPs (rs6165, rs6166, rs1394205) had no clinically significant impact on late follicular phase serum progesterone and estradiol levels in predicted normoresponders undergoing a GnRH antagonist protocol with a fixed daily dose of 150 IU rFSH. WHAT IS KNOWN ALREADY Previous studies have shown that late follicular phase serum progesterone and estradiol levels are significantly correlated with the magnitude of ovarian response. Several authors have proposed that individual variability in the response to ovarian stimulation (OS) could be explained by variants in FSHR. However, so far, the literature is scarce on the influence of this genetic variability on late follicular phase steroidogenic response. Our aim is to determine whether genetic variants in the FSHR gene could modulate late follicular phase serum progesterone and estradiol levels. STUDY DESIGN, SIZE, DURATION In this multicenter multinational prospective study conducted from November 2016 to June 2019, 366 patients from Vietnam, Belgium and Spain (166 from Europe and 200 from Asia) underwent OS followed by oocyte retrieval in a GnRH antagonist protocol with a fixed daily dose of 150 IU rFSH. All patients were genotyped for 3 FSHR SNPs (rs6165, rs6166, rs1394205) and had a serum progesterone and estradiol measurement on the day of trigger. PARTICIPANTS/MATERIALS, SETTING, METHODS Included patients were predicted normal responder women 1.50 ng/ml. MAIN RESULTS AND THE ROLE OF CHANCE The overall prevalence of PE was 15.8% (n = 58). No significant difference was found in the prevalence of PE in Caucasian and Asian patients (17.5% versus 14.5%). Estradiol levels on the day of trigger and the number of retrieved oocytes were significantly higher in patients with PE (4779 ± 6236.2 versus 3261 ± 3974.5 pg/ml, P = 0.003, and 16.1 ± 8.02 versus 13.5 ± 6.66, P = 0.011, respectively). Genetic model analysis, adjusted for patient age, body mass index, number of retrieved oocytes and continent (Asia versus Europe), revealed a similar prevalence of PE in co-dominant, dominant and recessive models for variants FSHR rs6166, rs6165 and rs1394205. No statistically significant difference was observed in the mean late follicular phase progesterone serum levels according to the genotypes of FSHR rs6166 (P = 0.941), rs6165 (P = 0.637) and rs1394205 (P = 0.114) in the bivariate analysis. Also, no difference was found in the genetic model analysis regarding mean late follicular phase progesterone levels across the different genotypes. Genetic model analysis has also revealed no statistically significant difference regarding mean estradiol levels on the day of trigger in co-dominant, dominant and recessive models for variants FSHR rs6166, rs6165 and rs1394205. Haplotype analysis revealed a statistically significant lower estradiol level on the day of trigger for rs6166/rs6165 haplotypes GA, AA and GG when compared to AG (respectively, estimated mean difference (EMD) −441.46 pg/ml (95% CI −442.47; −440.45), EMD −673.46 pg/ml (95% CI −674.26; −672.67) and EMD −582.10 pg/ml (95% CI −584.92; −579.28)). No statistically significant differences were found regarding the prevalence of PE nor late follicular phase progesterone levels according to rs6166/rs6165 haplotypes. LIMITATIONS, REASONS FOR CAUTION Results refer to a population of predicted normal responders treated with a normal/low fixed dose of 150 IU rFSH throughout the whole OS. Consequently, caution is needed before generalizing our results to all patient categories. WIDER IMPLICATIONS OF THE FINDINGS Based on our results, FSHR SNPs rs6165, rs6166 and rs1394205 do not have any clinically significant impact neither on late follicular phase serum progesterone nor on estradiol levels in predicted normal responders. These findings add to the controversy in the literature regarding the impact of individual genetic susceptibility in response to OS in this population. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by an unrestricted grant by Merck Sharp & Dohme (MSD, IISP56222). N.P.P. reports grants and/or personal fees from MSD, Merck Serono, Roche Diagnostics, Ferring International, Besins Healthcare, Gedeon Richter, Organon, Theramex and Institut Biochimique SA (IBSA). C.A. reports conference fees from Merck Serono, Medea and Event Planet. A.R.N., C.B., C.S., P.Q.M.M., H.T., C.B., N.L.V., M.T.H. and S.G. report no conflict of interests related to the content of this article. TRIAL REGISTRATION NUMBER NCT03007043.

Background: : Urine collection is a non-invasive self-sampling method offering the prospect of reaching women un (der)-screened for cervical cancer. The VALHUDES research framework was designed to address the lack of clinical accuracy data for high-risk (hr)HPV testing using urine samples. Objectives: : Here, we report on the analytical and clinical accuracy of hrHPV testing on first-void urine, collected at home, using an extended HPV genotyping assay. Study design: : Paired first-void urine (Colli-Pee with UCM, Novosanis; index test) and clinician-collected cervical samples (Cervex-Brush, Rovers in PreservCyt Solution, Hologic; comparator test) were collected from 492 women aged 19 to 72 years attending colposcopy (reference test, with histology if indicated) (VALHUDES; NCT03064087). Extended HPV genotyping was performed on paired samples with the BD Onclarity HPV Assay. Cut-offs defined for cervical samples were also applied for first-void urine. Results: : HrHPV testing in first-void urine was similarly sensitive for both CIN2+ (ratio 1.00; 95% CI: 0.93-1.07) and CIN3 (ratio 0.98; 95% CI: 0.88-1.08), and marginally less specific for 2 (ratio 0.92; 95% CI: 0.84-0.996) compared to cervical samples. HPV test agreement between sample pairs expressed as Cohen's Kappa (kappa) was moderate to excellent for overall hrHPV and individual genotypes (or groups) (kappa=0.56-0.85). Conclusions: : BD Onclarity HPV Assay on first-void urine has similar clinical sensitivity and somewhat lower specificity to detect cervical precancer to testing on clinician-collected cervical samples.

The microbiome of the reproductive tract has been associated with (sub)fertility and it has been suggested that dysbiosis reduces success rates and pregnancy outcomes. The endome-trial microbiome is of particular interest given the potential impact on the embryo implanta-tion. To date, all endometrial microbiome studies have applied a metagenomics approach. A se-quencing-based technique, however, has its limitations, more specifically in adequately explor-ing low-biomass settings, such as intra-uterine/endometrial samples. In this proof-of-concept study, we demonstrate the applicability of culturomics, a high-throughput culturing approach, to investigate the endometrial microbiome. Ten subfertile women undergoing diagnostic hys-teroscopy and endometrial biopsy, as part of their routine work-up at Brussels IVF, were includ-ed after their informed consent. Biopsies were used to culture microbiota for up to 30 days in multiple aerobic and anaerobic conditions. Subsequent WASPLab®-assisted culturomics enabled a standardized methodology. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) or 16S rRNA sequencing was applied to identify all of bacterial and fungal isolates. Eighty-three bacterial and two fungal species were identified. The detected species were in concordance with previously published metagenomics-based endometrial mi-crobiota analyses as 77 (91%) of them belonged to previously described genera. Nevertheless, highlighting the added value of culturomics to identify most isolates at the species level, 53 (62.4%) of the identified species were described in the endometrial microbiota for the first time. This study shows the applicability and added value of WASPLab®-assisted culturomics to inves-tigate the low biomass endometrial microbiome at a species level.

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

Background Interventions to reach women who do not participate regularly in screening may reduce the risk of cervical cancer. Self-collection of a vaginal specimen has been shown to increase participation. The relative clinical accuracy of human papillomavirus (HPV) testing on first-void urine (with Colli-Pee) and on vaginal self-samples versus on cervical clinician-collected samples is being investigated in the VALHUDES trial. The current study assesses attitudes and experiences regarding self-sampling among women enrolled in VALHUDES. Methods Questionnaires from 515 women (age 25–64 years [N = 498]; N = 10], age ≥ 65 [N = 3], enrolled between December 2017 - January 2020) referred to colposcopy because of previous cervical abnormalities and enrolled in VALHUDES (NCT03064087) were analysed. Results Of the 515 participants, nearly all women confirmed that self-sampling may help in reaching under-screened women (93%). Nevertheless, 44% of the participants stated before starting collection that a clinician-collected sample is more effective than a self-collected sample. After self-sampling, the large majority of women (> 95%) declared that instructions for self-collection were clear, that collection was easy, and that they were confident about having performed the procedure correctly, for both urine and vaginal collection. However, a proportion of women found self-sampling unpleasant (9.5% [49/515] for urine collection; 18.6% [96/515] and 15.5% [80/515] for vaginal sampling with cotton swabs or plastic brushes, respectively). For their next screening round, 57% would prefer self-sampling whereas 41% opted for collection by a clinician. Among women preferring self-sampling, 53% would choose for urine collection, 38% for vaginal self-collection and 9% had no preference. Age did not modify preferences. Conclusion We conclude that both urine and vaginal self-sampling are well accepted by women, with a preference for urine sampling. Although the large majority of women are confident in their ability to perform self-sampling, four to five over ten women preferred specimen collection by a clinician. Trial registration The study VALHUDES was registered in ClinicalTrials.gov (identifier: NCT03064087).

Purpose: Although a robust physiological rationale supports follicle stimulating hormone (FSH) use in male idiopathic infertility, useful biomarkers to evaluate its efficacy are not available. Thus, the primary aim of the study was to evaluate if testosterone serum levels are related to sperm DNA fragmentation (sDF) index change after FSH administration. The secondary aim was to confirm sDF index validity as a biomarker of FSH administration effectiveness in male idiopathic infertility. Methods: A retrospective, post-hoc re-analysis was performed on prospectively collected raw data of clinical trials in which idiopathic infertile men were treated with FSH and both testosterone serum levels and sDF were reported. Results: Three trials were included, accounting for 251 patients. The comprehensive analysis confirmed FSH’s beneficial effect on spermatogenesis detected in each trial. Indeed, an overall significant sDF decrease (p < 0.001) of 20.2% of baseline value was detected. Although sDF resulted to be unrelated to testosterone serum levels at baseline, a significant correlation was highlighted after three months of FSH treatment (p = 0.002). Moreover, testosterone serum levels and patients’ age significantly correlated with sDF (p = 0.006). Dividing the cohort into responders/not responders to FSH treatment according to sDF change, the FSH effectiveness in terms of sDF improvement was related to testosterone and male age (p = 0.003). Conclusion: Exogenous FSH administration in male idiopathic infertility is efficient in reducing sDF basal levels by about 20%. In terms of sDF reduction, 59.2% of the patients treated were FSH-responders. After three months of FSH administration, a significant inverse correlation between sDF and testosterone was detected, suggesting an association between the FSH-administration-related sDF improvement and testosterone serum levels increase. These observations lead to the hypothesis that FSH may promote communications or interactions between Sertoli cells and Leydig cells.

RESEARCH QUESTION: Does ageing affect endometrial stromal cell function and gene expression involved in decidualization? DESIGN: Stromal cells were isolated and cultured (98% purity) from pipelle endometrial biopsies obtained from female donors, at the proliferative phase of the menstrual cycle. Initially, 28 samples were collected (age range 25-46 years). These samples were divided into two groups: 25-35 years and 36-46 years. Cell proliferation assays were carried out to determine changes in stromal cell proliferation, in vitro, between the two groups. In addition, mRNA and protein expression of decidualization factors, BMP2 and STAT3, were analysed for the same age groups and samples using real-time polymerase chain reaction and western blot analysis, respectively. Finally, another 12 pipelle endometrial biopsies (age range 25-46 years) were used in separate in-vitro decidualization experiments. The mRNA expression of decidualization markers, prolactin and IGFBP-1 were analysed in cultured stromal cells after adding 8-bromo-cAMP. RESULTS: A statistically significant decrease was observed in stromal cell proliferation with increasing age (P = 0.0006). Messenger RNA expression of BMP2 and STAT3 were found to decrease (P = 0.0167; P = 0.0037, respectively) in the older age group. In addition, BMP2 and STAT3 protein expression between the samples analysed changed significantly (P = 0.0085; P = 0.0463, respectively) with increasing age. In-vitro induced decidualization experiments showed significant changes in stromal cell mRNA expression of decidualization markers (IGFBP1 and prolactin) between different age groups. CONCLUSION: Ageing affected endometrial cell function and gene expression. Changes in cell function and expression of associated molecules that differ in the ageing endometrium can help understand the causes of infertility.

IntroductionExisting randomised controlled trials (RCTs) comparing a freeze-all embryo transfer strategy and a fresh embryo transfer strategy have shown conflicting results. A freeze-all or a fresh transfer policy may be preferable for some couples undergoing in-vitro fertilisation (IVF), but it is unclear which couples would benefit most from each policy, how and under which protocols. Therefore, we plan a systematic review and individual participant data meta-analysis of RCTs comparing a freeze-all and a fresh transfer policy.Methods and analysisWe will search electronic databases (Medline, Embase, PsycINFO and CENTRAL) and trial registries (ClinicalTrials.gov and the International Clinical Trials Registry Platform) from their inception to present to identify eligible RCTs. We will also check reference lists of relevant papers. The search was performed on 23 September 2020 and will be updated. We will include RCTs comparing a freeze-all embryo transfer strategy and a fresh embryo transfer strategy in couples undergoing IVF. The primary outcome will be live birth resulting from the first embryo transfer. All outcomes listed in the core outcome set for infertility research will be reported. We will invite the lead investigators of eligible trials to join the Individual participant data meta-analysis of trials comparing frozen versus fresh embryo transfer strategy (INFORM) collaboration and share the deidentified individual participant data (IPD) of their trials. We will harmonise the IPD and perform a two-stage meta-analysis and examine treatment–covariate interactions for important baseline characteristics.Ethics and disseminationThe study ethics have been granted by the Monash University Human Research Ethics Committee (Project ID: 30391). The findings will be disseminated via presentations at international conferences and publication in peer-reviewed journals.PROSPERO registration numberCRD42021296566.

The use of frozen embryo transfer cycles has exponentially increased in the last few years. Optimization of endometrial preparation protocols before frozen embryo transfer is mandatory to further improve pregnancy outcomes. This review will focus on the existing literature with regard to the different available endometrial preparation protocols and their impact on pregnancy outcomes. More specifically, we will focus on programmed, natural, and stimulated frozen embryo transfer cycles. The studies performed on this topic are generally of low quality, and only a few well-performed randomized controlled trials have been published. To date, no strong evidence is available to support the use of 1 preparation method over another in terms of pregnancy outcomes. However, robust data have shown a clearly protective effect of natural frozen embryo transfer cycles against long-term obstetric complications, mainly hypertensive disorders of pregnancy and large for gestational age infants. The introduction of individualized luteal phase support in different endometrial preparation protocols is actually gaining a lot of attention and requires further investigation.

Background: In this study, we evaluated accuracy of HPV testing on self-samples versus clinician-taken samples through the VALHUDES protocol. VALHUDES was designed as a diagnostic test accuracy study, where women referred to colposcopy collected self-samples followed by clinician-taken cervical samples. Methods: Four hundred eighty-five women recruited in five colposcopy clinics (median age = 40 years; IQR, 31–49) with valid results for all specimens were included in the main analysis: 230 vaginal self-samples were collected with Evalyn Brush and 255 with Qvintip. Cervical samples were taken by the gynecologist with the Cervex-Brush. HPV testing was performed with BD Onclarity HPV assay (Onclarity). Colposcopy and histology were used as the reference standard for accuracy estimation. Results: The sensitivity for CIN2+ on vaginal self-samples overall was not different from cervical samples (ratio = 0.96; 95% CI, 0.90–1.03), whereas specificity was significantly higher (ratio = 1.09; 95% CI, 1.02–1.16). However, the relative accuracy (self- vs. clinician sampling) differed by vaginal collection device: relative sensitivity and specificity ratios of 1.00 (95% CI, 0.94–1.06) and 1.15 (95% CI, 1.05–1.25), respectively for Evalyn-Brush; 0.91 (95% CI, 0.79–1.04) and 1.03 (95% CI, 0.95–1.13), respectively for Qvintip. Conclusions: Clinical accuracy of BD Onclarity HPV assay on vaginal self-samples was not different from cervical samples. Impact: VALHUDES study showed that HPV testing with Onclarity HPV on vaginal self-samples is similarly sensitive compared with cervical specimens. However, differences in accuracy by self-sampling devices, although not significant, were noted. Onclarity HPV testing on vaginal self-samples following validated collection and handling procedures may be used in primary cervical cancer screening.

Intracytoplasmic sperm injection (ICSI), developed 30 years ago in our Center, allowed men with low to extremely low sperm quality to become the genetic father of their child. Overall, ICSI is now applied in up to 70% of all ART cycles and has resulted in the birth of millions of children worldwide. ICSI circumvents natural sperm selection mechanisms and concerns related to transgenerational inheritance of male infertility following ICSI could not be fully answered till today, given the young age of the offspring born after ICSI. Since genetic abnormalities account for one third of all cases of male factor infertility, it is essential to identify these genetic abnormalities because of the potential risk of their transmission to the offspring through the use of ART. As ICSI is nowadays performed even in couples with normal semen parameters, the disentanglement of procedure-related and parental background-related factors on the health of the offspring is important. Due to the safety concerns regarding the health of children born after ART, a longitudinal follow-up program was set up in our Center in the 80’s, resulting in data of more than 50 000 pregnancies. To date, more than 25 000 children are conceived after ICSI in our Center. We have not only the worldwide eldest cohort of ICSI offspring but also a uniform cohort of children and young adults conceived by ICSI because of (severe) male factor infertility. We present the results of these prospective studies with focus on the reproductive health in ICSI children between 8 and 22 years and compare these with available data from others. First of all, we will show data on the gonadal function assessed both clinically and by means of biomarkers at pre-pubertal and pubertal ages in ICSI boys. Secondly, we will present findings on the reproductive status of young adult men and women conceived after ICSI between 1992 and 1996: results of hormonal work-up, physical examination and semen parameters for ICSI men and results of hormone levels and antral follicle counts for ICSI women. Finally, we will discuss the findings in these cohorts of children and young adults conceived because of male infertility in view of the available literature regarding reproductive function in offspring conceived following ICSI.

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PURPOSE: To investigate whether treatment with commercially available ready-to-use A23187 ionophore (GM508-CultActive) improves embryo development outcome in patients with a history of embryo developmental problems. METHODS: This is a uni-center prospective study in which sibling oocytes of patients with embryos of poor quality on day 5 in the previous cycle were treated or not with CultActive. RESULTS: Two hundred forty-seven metaphase II (MII) oocytes from 19 cycles performed between 2016 and 2019 were included in the study. After ICSI, the sibling oocytes were assigned to the treatment group or to the control group, following an electronically generated randomization list. A number of 122 MII were treated with CultActive and 125 MII had no treatment and were assigned to the control group. No difference in fertilization rate (p = 0.255) or in the capacity of embryos to reach good quality on day 5 (p = 0.197) was observed between the two groups. The utilization rates defined as the number of embryos transferred or cryopreserved per mature oocyte (p = 0.438) or per fertilized oocytes (p = 0.299) were not significantly different between the treated group and the control group. CONCLUSION: The results of the current study do not support the use of CultActive in cases with embryo developmental problems.

Parasitic leiomyomas are a rare subtype of subserosal uterine leiomyomas and are mostly found incidentally. Diagnosis is challenging and treatment consists out of complete surgical resection. Cases are few in women without history of uterine surgery. In this extraordinary case, a 56-year-old menopausal female suffering from backache and abdominal swelling, was suspected to have a huge malignant ovarian tumor. She underwent a resection of the mass along with hysterectomy and bilateral salpingo-oöphorectomy. Histopathology revealed a gigantic leiomyoma of 19.1 kg in the broad ligament. This unique case suggests taking this diagnosis into account in future clinical cases presenting with large abdominal masses even without previous uterine surgery.

PURPOSE: To report the first successful application of in vitro maturation (IVM) of oocytes resulting in live births in two anovulatory women who had suffered oophorectomy following ovarian torsion after stimulation with gonadotropins. METHODS: Data abstraction was performed from medical records of two subfertile women with excessive functional ovarian reserve. Both women had previously received gonadotropins for ovulation induction or ovarian stimulation, resulting in ovarian torsion. They were offered IVM of oocytes retrieved from antral follicles after mild ovarian stimulation, insemination of mature oocytes using ICSI, and embryo transfer. Outcome measures were the incidence of complications and live birth after fertility treatment. RESULTS: Transvaginal retrieval of cumulus-oocyte complexes from a unique ovary was conducted. One patient had a singleton live birth after vitrified-warmed embryo transfer in the second IVM cycle. The other patient had a singleton live birth after transfer of a fresh blastocyst in her first IVM cycle. CONCLUSIONS: Although approaches have been developed to prevent ovarian hyperstimulation syndrome (OHSS) and to increase the safety profile of fertility treatment in predicted high responders, women with an excessive functional ovarian reserve may have a non-negligible risk of ovarian torsion. For these patients, IVM should be considered as a safer alternative approach.

RESEARCH QUESTION: How do cumulative live birth rates (CLBR), cumulative multiple live birth rates (CMLBR) and dropout rates over six IVF and intracytoplasmic sperm injection (ICSI) cycles change over time? DESIGN: Prospective longitudinal cohort (n = 16,073 patients; 48,946 cycles) starting a first fresh assisted reproductive technology cycle between 1 January 2014 and 31 December 2016, with follow-up until 31 December 2017. Outcomes between the periods 2014-2017 and 2009-2012 were compared. RESULTS: Conservative estimates of CLBR after six complete cycles were significantly higher in women younger than 35 years after every cycle: one to three, adjusted P-value [p adj] < 0.0001; four, p = 0.01; five, p adj = 0.03; six, p adj = 0.04) and after the first cycle in women aged 35-37 years (p adj = 0.04) in 2014-2017 versus 2009-2012. For an optimal estimate, the CLBR was significantly higher after the first three cycles in women younger than 35 years (all p adj < 0.0001) and after the first cycle in women aged 35-37 years (p adj = 0.04). The CMLBR rate decreased from 5.1% ± 0.19 (SE) to 4.1% ± 0.16 for the conservative estimate and from 8.6% ±0.37 (SE) to 6.7% ± 0.30 for the optimal estimate after six complete cycles for the whole cohort. Dropout rates of complete cycles were 26.5% 29.4%, 33.4%, 38.9% and 47.3% after the first to fifth cycle, respectively. Compared with 2009-2012, the dropout rate in the current period was significantly higher for the first (P < 0.0001) and second (P = 0.0124) cycle. CONCLUSION: Over six complete IVF/ICSI cycles, CLBR and dropout rates increased and multiple live birth rates decreased when 2014-2017 was compared with 2009-2012. ispartof: REPRODUCTIVE BIOMEDICINE ONLINE vol:42 issue:4 pages:717-724 ispartof: location:Netherlands status: published

Patients with childhood, adolescent, and young adult cancer who will be treated with gonadotoxic therapies are at increased risk for infertility. Many patients and their families desire biological children but effective communication about treatment-related infertility risk and procedures for fertility preservation does not always happen. The PanCareLIFE Consortium and the International Late Effects of Childhood Cancer Guideline Harmonization Group reviewed the literature and developed a clinical practice guideline that provides recommendations for ongoing communication methods for fertility preservation for patients who were diagnosed with childhood, adolescent, and young adult cancer at age 25 years or younger and their families. Moreover, the guideline panel formulated considerations of the ethical implications that are associated with these procedures. Grading of Recommendations Assessment, Development and Evaluation methodology was used to grade the evidence and recommendations. In this clinical practice guideline, existing evidence and international expertise are combined to develop transparent recommendations that are easy to use to facilitate ongoing communication between health-care providers and patients with childhood, adolescent, and young adult cancer who might be at high risk for fertility impairment and their families.

PURPOSE: Does cell loss (CL) after vitrification and warming (V/W) of day 3 embryos have an impact on live birth rate (LBR) and neonatal outcomes? METHOD: This retrospective analysis includes cleavage stage day 3 embryos vitrified/warmed between 2011 and 2018. Only single vitrified/warmed embryo transfers were included. Pre-implantation genetic screening, oocyte donation, and age banking were excluded from the analysis. The sample was divided into two groups: group A (intact embryo after warming) and group B (≤ 50% blastomere loss after warming). RESULTS: On the total embryos (n = 2327), 1953 were fully intact (83.9%, group A) and 374 presented cell damage (16.1%, group B). In group B, 62% (232/374) of the embryos had lost only one cell. Age at cryopreservation, cause of infertility, insemination procedure, and semen origin were comparable between the two groups. The positive hCG rate (30% and 24.3%, respectively, for intact vs CL group, p = 0.028) and LBR (13.7% and 9.4%, respectively, for intact vs CL group, p = 0.023) per warming cycle were significantly higher for intact embryos. However, LBR per positive hCG was equivalent between intact and damaged embryos (45.6% vs 38.5%, respectively, p = 0.2). Newborn measurements (length, weight, and head circumference at birth) were comparable between the two groups. Multivariate logistic regression showed that the presence of CL is not predictive for LB when adjusting for patients' age. CONCLUSIONS: LBR is significantly higher after transfer of an intact embryo compared to an embryo with CL after warming; however, neonatal outcomes are comparable between the two groups.