Association between the length of in vitro embryo culture, mode of ART, and the initial endogenous hCG rise in ongoing singleton pregnancies.

STUDY QUESTION: Is there an association between the length of in vitro culture, mode of ART and the initial endogenous hCG rise, in cycles with a foetal heartbeat after single embryo transfer (ET) and implantation? SUMMARY ANSWER: Both the length of in vitro culture and the mode of ART have an impact on the initial endogenous rise in hCG in singleton pregnancies. WHAT IS KNOWN ALREADY: Different factors have been identified to alter the kinetics of hCG in pregnancies. Current studies show conflicting results regarding the kinetics of hCG after different types of ART (fresh vs frozen ET (FET)), the inclusion or not of preimplantation genetic testing (PGT), and the length of time in in vitro culture. STUDY DESIGN, SIZE, DURATION: This was a multicentre cohort study, using prospectively collected data derived from 4938 women (5524 treatment cycles) undergoing IUI (cycles, n = 608) or ART (cycles, n = 4916) treatments, resulting a in singleton ongoing pregnancy verified by first-trimester ultrasound scan. Data were collected from the Danish Medical Data Centre, used by the three participating Danish public fertility clinics at Copenhagen University hospitals: Herlev Hospital, Hvidovre Hospital, and Rigshospitalet, from January 2014 to December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: The fresh ET cycles included cleavage-stage (2 or 3 days in vitro) and blastocyst (5 days in vitro) transfers. FET cycles included cleavage-stage (3 days in vitro before cryopreservation) or blastocyst (5 or 6 days in vitro before cryopreservation) transfers. The IUI cycles represented no time in vitro. To attain a comparable interval for serum-hCG (s-hCG), the ovulation induction time was identical: 35-37 h before oocyte retrieval or IUI. The conception day was considered as: the insemination day for pregnancies conceived after IUI, the oocyte retrieval day for fresh ET, or the transfer day minus 3 or 5 as appropriate for FET of Day 3 or 5 embryos. Multiple linear regression analysis was used, including days post-conception for the hCG measurement as a covariate, and was adjusted for the women's age, the cause of infertility, and the centre. For FET, a sensitivity analysis was used to adjust for endometrial preparation. MAIN RESULTS AND THE ROLE OF CHANCE: The study totally includes 5524 cycles: 2395 FET cycles, 2521 fresh ET cycles, and 608 IUI cycles. Regarding the length of in vitro culture, with IUI as reference (for no time in in vitro culture), we found a significantly lower s-hCG in pregnancies achieved after fresh ET (cleavage-stage ET or blastocyst transfer). S-hCG was 18% (95% CI: 13-23%, P < 0.001) lower after fresh cleavage-stage ET, and 23% (95% CI: 18-28%, P < 0.001) lower after fresh blastocyst transfer compared to IUI. In FET cycles, s-hCG was significantly higher after blastocyst transfers compared to cleavage-stage FET, respectively, 26% (95% CI: 13-40%, P < 0.001) higher when cryopreserved on in vitro Day 5, and 14% (95% CI: 2-26%, P = 0.02) higher when cryopreserved on in vitro Day 6 as compared to Day 3. Regarding the ART treatment type, s-hCG after FET blastocyst transfer (Day 5 blastocysts) cycles was significantly higher, 33% (95% CI: 27-45%, P < 0.001), compared to fresh ET (Day 5 blastocyst), while there was no difference between cleavage-stage FET (Days 2 + 3) and fresh ET (Days 2 + 3). S-hCG was 12% (95% CI: 4-19%, 0.005) lower in PGT FET (Day 5 blastocysts) cycles as compared to FET cycles without PGT (Day 5 blastocysts). LIMITATIONS, REASONS FOR CAUTION: The retrospective design is a limitation which introduces the risk of possible bias and confounders such as embryo score, parity, and ovarian stimulation. WIDER IMPLICATIONS OF THE FINDINGS: This study elucidates how practices in medically assisted reproduction treatment are associated with the hCG kinetics, underlining a potential impact of in vitro culture length and mode of ART on the very early embryo development and implantation. The study provides clinicians knowledge that the type of ART used may be relevant to take into account when evaluating s-hCG for the prognosis of the pregnancy. STUDY FUNDING/COMPETING INTEREST(S): No funding was received for this study. AP has received consulting fees, research grants, or honoraria from the following companies: Preglem, Novo Nordisk, Ferring Pharmaceuticals, Gedeon Richter, Cryos, Merck A/S, and Organon. AZ has received grants and honoraria from Gedeon Richter. NLF has received grants from Gedeon Richter, Merck A/S, and Cryos. MLG has received honoraria fees or research grants from Gedeon Richter, Merck A/S, and Cooper Surgical. CB has received honoraria from Merck A/S. MB has received research grants and honoraria from IBSA. MPR, KM, and PVS all report no conflicts of interest. TRIAL REGISTRATION NUMBER: The study was registered and approved by the Danish Protection Agency, Capital Region, Denmark (Journal-nr.: 21019857). No approval was required from the regional ethics committee according to Danish law.

Predictive performance of peritoneal fluid in the pouch of Douglas measured five days after oocyte pick-up in predicting severe late-onset OHSS: A secondary analysis of a randomized trial.

OBJECTIVES: To investigate if the amount of peritoneal fluid (PF) in the Pouch of Douglas at oocyte pick-up (OPU) or OPU + 5 days predict severe late-onset ovarian hyperstimulation syndrome (OHSS) in women undergoing ovarian stimulation for assisted reproductive technology (ART). STUDY DESIGN: A secondary analysis of a dual-centre RCT on 1050 women referred for their first ART treatment in two public fertility clinics in Denmark and randomized 1:1 to GnRH-antagonist or GnRH-agonist protocol. All women from the two arms who were examined on day of OPU and OPU + 5 days were included in this study (n = 940). The ability of PF in the pouch of Douglas to predict severe late-onset OHSS was assessed by multivariate logistic regression analyses and receiver operator characteristics (ROC) curve analyses and compared with other known predictors of OHSS. The final models were cross-validated by the leave-one-out method to assess the models' generalizability. RESULTS: A total of 28 (3%) women developed severe late-onset OHSS. PF in the pouch of Douglas measured on OPU + 5 days predicted severe late-onset OHSS. The optimal cut-off value was 17.5 mm at OPU + 5 days with a 61% sensitivity and 71% specificity (Area under the curve = 0.70 95% CI 0.61-0.80). PF on the day of OPU was not predictive of late on-set OHSS as the adjusted multivariate logistic regression analyses showed insignificant results. CONCLUSION: Although PF in the pouch of Douglas could predict late-onset severe OHSS, the low sensitivity underlines that it is not useful as a sole marker to decide whether to perform blastocyst transfer or to use a freeze-all strategy.

Developmental stage and morphology of the competent blastocyst are associated with sex of the child but not with other obstetric outcomes: a multicenter cohort study.

STUDY QUESTION: Are transfer day, developmental stage and morphology of the competent blastocyst in pregnancies leading to live birth associated with preterm birth, birthweight, length at birth and sex of the child? SUMMARY ANSWER: A high score in blastocyst developmental stage and in trophectoderm (TE) showed a significant association with the sex of the child, while no other associations with obstetric outcomes were observed. WHAT IS KNOWN ALREADY: The association between blastocyst assessment scores and obstetric outcomes have been reported in small single-center studies and the results are conflicting. STUDY DESIGN, SIZE, DURATION: Multicenter historical cohort study based on exposure data (transfer day (blastocyst developmental stage reached by Day 5 or Day 6)) blastocyst developmental stage (1-6) and morphology (TE and inner cell mass (ICM): A, B, C)) and outcome data (preterm birth, birthweight, length at birth, and sex of the child) from women undergoing single blastocyst transfer resulting in a singleton pregnancy and live birth. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from 16 private and university-based facilities for clinical services and research were used. A total of 7246 women, who in 2014-2018 underwent fresh-embryo transfer with a single blastocyst or frozen-thawed embryo transfer (FET) with a single blastocyst resulting in a singleton pregnancy were identified. Linking to the Danish Medical Birth Registry resulted in a total of 4842 women with a live birth being included. Cycles with pre-implantation genetic testing and donated gametes were excluded. The analyses were adjusted for female age (n = 4842), female BMI (n = 4302), female smoking (n = 4290), parity (n = 4365), infertility diagnosis (n = 4765), type of treatment (n = 4842) and center (n = 4842); some analyses additionally included gestational age (n = 4368) and sex of the child (n = 4833). MAIN RESULTS AND THE ROLE OF CHANCE: No statistically significant associations between blastocyst assessment scores (transfer day, developmental stage, TE, ICM) and preterm birth (8.3%) or birthweight (mean 3461.7 g) were found. The adjusted association between blastocysts with a TE score of C and a TE score of A and length at birth (mean 51.6 cm) were statistically significant (adjusted mean difference 0.4 cm (95% CI: 0.02; 0.77)). Blastocysts transferred with developmental stage score 5 compared to blastocysts transferred with score 3 had a 34% increased probability of being a boy (odds ratio (OR) 1.34 (95% CI: 1.09; 1.64). Further, TE score B blastocysts compared to TE score A blastocysts had a 31% reduced probability of being a boy (OR 0.69 (95% CI: 0.60; 0.80)). LIMITATIONS, REASONS FOR CAUTION: It is possible that some residual confounding remains. WIDER IMPLICATIONS OF THE FINDINGS: Blastocyst selection during ART does not appear to introduce any negative effects on obstetric outcome. Therefore, clinicians and patients can be reassured that the assessment scores of the selected blastocyst will not in themselves pose a risk of preterm birth or affect birthweight and the length at birth. STUDY FUNDING/COMPETING INTEREST(S): Unrestricted grant from Gedeon Richter Nordics AB, Sweden. None of the authors have any competing interest to declare. TRIAL REGISTRATION NUMBER: N/A.

SARS-CoV-2 in first trimester pregnancy: a cohort study.

STUDY QUESTION: Does maternal infection with severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) in first trimester pregnancy have an impact on the fetal development as measured by nuchal translucency thickness and pregnancy loss? SUMMARY ANSWER: Nuchal translucency thickness at the first trimester scan was not significantly different in pregnant women with versus without SARS-CoV-2 infection in early pregnancy and there was no significantly increased risk of pregnancy loss in women with SARS-CoV-2 infection in the first trimester. WHAT IS KNOWN ALREADY: Pregnant women are more vulnerable to viral infections. Previous coronavirus epidemics have been associated with increased maternal morbidity, mortality and adverse obstetric outcomes. Currently, no evidence exists regarding possible effects of SARS-CoV-2 in first trimester pregnancies. STUDY DESIGN, SIZE, DURATION: Cohort study of 1019 women with a double test taken between 17 February and 23 April 2020, as a part of the combined first trimester risk assessment, and 36 women with a first trimester pregnancy loss between 14 April and 21 May 2020, prior to the double test. The study period was during the first SARS-CoV-2 epidemic wave in Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cohort 1 included pregnant women with a double test taken within the study period. The excess serum from each double test was analyzed for SARS-CoV-2 antibodies. Results were correlated to the nuchal translucency thickness and the number of pregnancy losses before or at the time of the first trimester scan. Cohort 2 included women with a pregnancy loss before the gestational age for double test sample. Serum from a blood test taken the day the pregnancy loss was identified was analyzed for SARS-CoV-2 antibodies. The study was conducted at a public university hospital serving ∼12% of pregnant women and births in Denmark. All participants in the study provided written informed consent. MAIN RESULTS AND THE ROLE OF CHANCE: Eighteen (1.8%) women had SARS-CoV-2 antibodies in the serum from the double test suggestive of SARS-CoV-2 infection in early pregnancy. There was no significant difference in nuchal translucency thickness for women testing positive for previous SARS-CoV-2 infection (n = 16) versus negative (n = 966) (P = 0.62). There was no significantly increased risk of pregnancy loss for women with antibodies (n = 1) (OR 3.4, 0.08-24.3 95% CI, P = 0.27). None of the women had been hospitalized due to SARS-CoV-2 infection. None of the women with pregnancy loss prior to the double test (Cohort 2) had SARS-CoV-2 antibodies. LIMITATIONS, REASONS FOR CAUTION: These results may only apply to similar populations and to patients who do not require hospitalization due to SARS-CoV-2 infection. A limitation of the study is that only 1.8% of the study population had SARS-CoV-2 antibodies suggestive of previous infection. WIDER IMPLICATION OF THE FINDINGS: Maternal SARS-CoV-2 infection had no effect on the nuchal translucency thickness and there was no significantly increased risk of pregnancy loss for women with SARS-CoV-2 infection in first trimester pregnancy. Evidence concerning COVID-19 in pregnancy is still limited. These data indicate that infection with SARS-CoV-2 in not hospitalized women does not pose a significant threat in first trimester pregnancies. Follow-up studies are needed to establish any risk to a fetus exposed to maternal SARS-CoV-2 infection. STUDY FUNDING/COMPETING INTEREST(S): Prof. H.S.N. and colleagues received a grant from the Danish Ministry of Research and Education for research of COVID-19 among pregnant women. The Danish government was not involved in the study design, data collection, analysis, interpretation of data, writing of the report or decision to submit the paper for publication. A.I., J.O.-L., J.B.-R., D.M.S., J.E.-F. and E.R.H. received funding from a Novo Nordisk Foundation (NNF) Young Investigator Grant (NNF15OC0016662) and a Danish National Science Foundation Center Grant (6110-00344B). A.I. received a Novo Scholarship. J.O.-L. is funded by an NNF Pregraduate Fellowship (NNF19OC0058982). D.W. is funded by the NNF (NNF18SA0034956, NNF14CC0001, NNF17OC0027594). A.M.K. is funded by a grant from the Rigshospitalet's research fund. H.S.N. has received speaker's fees from Ferring Pharmaceuticals, Merck Denmark A/S and Ibsa Nordic (outside the submitted work). N.l.C.F. has received a grant from Gedeon Richter (outside the submitted work). A.M.K. has received speaker's fee from Merck (outside the submitted work). The other authors did not report any potential conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Rare genetic variants suggest dysregulation of signaling pathways in low- and high-risk patients developing severe ovarian hyperstimulation syndrome.

PURPOSE: To investigate if rare gene variants in women with severe ovarian hyperstimulation syndrome (OHSS) provide clues to the mechanisms involved in the syndrome. METHODS: Among participants in a prospective randomized study (Toftager et al. 2016), six women with predicted low and six women with predicted high risk of OHSS developing severe OHSS (grades 4 and 5, Golan classification) were selected. In the same cohort, six plus six matched controls developing no signs of OHSS (Golan grade 0) were selected. Whole-exome sequencing was performed. Analysis using a predefined in silico OHSS gene panel, variant filtering, and pathway analyses was done. RESULTS: We found no convincing monogenetic association with the development of OHSS using the in silico gene panel. Pathway analysis of OHSS variant lists showed substantial overlap in highly enriched top pathways (p value range p < 0.0001 and p > 9.8E-17) between the low- and high-risk group developing severe OHSS, i.e., "the integrin-linked kinase (ILK) signaling pathway" and the "axonal guidance signaling pathway," both being connected to vasoactive endothelial growth factor (VEGF) and endothelial function. CONCLUSION: Rare variants in OHSS cases with two distinct risk profiles enrich the same signaling pathways linked to VEGF and endothelial function. Clarification of the mechanism as well as potentially defining genetic predisposition of the high vascular permeability is important for future targeted treatment and prevention of OHSS; the potential roles of ILK signaling and the axonal guidance signaling need to be validated by functional studies.

A distinctive epigenetic ageing profile in human granulosa cells.

STUDY QUESTION: Does women's age affect the DNA methylation (DNAm) profile differently in mural granulosa cells (MGCs) from other somatic cells? SUMMARY ANSWER: Accumulation of epimutations by age and a higher number of age-related differentially methylated regions (DMR) in MGCs were found compared to leukocytes from the same woman, suggesting that the MGCs have a distinctive epigenetic profile. WHAT IS KNOWN ALREADY: The mechanisms underlying the decline in women's fertility from the mid-30s remain to be fully elucidated. The DNAm age of many healthy tissues changes predictably with and follows chronological age, but DNAm age in some reproductive tissues has been shown to depart from chronological age (older: endometrium; younger: cumulus cells, spermatozoa). STUDY DESIGN, SIZE, DURATION: This study is a multicenter cohort study based on retrospective analysis of prospectively collected data and material derived from healthy women undergoing IVF or ICSI treatment following ovarian stimulation with antagonist protocol. One hundred and nineteen women were included from September 2016 to June 2018 from four clinics in Denmark and Sweden. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples were obtained from 118 healthy women with varying ovarian reserve status. MGCs were collected from 63 of the 119 women by isolation from pooled follicles immediately after oocyte retrieval. DNA from leukocytes and MGCs was extracted and analysed with a genome-wide methylation array. Data from the methylation array were processed using the ENmix package. Subsequently, DNAm age was calculated using established and tailored age predictors and DMRs were analysed with the DMRcate package. MAIN RESULTS AND ROLE OF CHANCE: Using established age predictors, DNAm age in MGCs was found to be considerable younger and constant (average: 2.7 years) compared to chronological age (average: 33.9 years). A Granulosa Cell clock able to predict the age of both MGCs (average: 32.4 years) and leukocytes (average: 38.8 years) was successfully developed. MGCs differed from leukocytes in having a higher number of epimutations (P = 0.003) but predicted telomere lengths unaffected by age (Pearson's correlation coefficient = -0.1, P = 0.47). DMRs associated with age (age-DMRs) were identified in MGCs (n = 335) and in leukocytes (n = 1) with a significant enrichment in MGCs for genes involved in RNA processing (45 genes, P = 3.96 × 10-08) and gene expression (152 genes, P = 2.3 × 10-06). The top age-DMRs included the metastable epiallele VTRNA2-1, the DNAm regulator ZFP57 and the anti-Müllerian hormone (AMH) gene. The apparent discordance between different epigenetic measures of age in MGCs suggests that they reflect difference stages in the MGC life cycle. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: No gene expression data were available to associate with the epigenetic findings. The MGCs are collected during ovarian stimulation, which may influence DNAm; however, no correlation between FSH dose and number of epimutations was found. WIDER IMPLICATIONS OF THE FINDINGS: Our findings underline that the somatic compartment of the follicle follows a different methylation trajectory with age than other somatic cells. The higher number of epimutations and age-DMRs in MGCs suggest that their function is affected by age. STUDY FUNDING/COMPETING INTEREST(S): This project is part of ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS, the Danish National Research Foundation and the European Research Council. The authors declare no conflict of interest.

Patients' attitudes and preferences towards a freeze-all strategy in ART treatment.

STUDY QUESTION: What are the attitudes towards different aspects of a freeze-all strategy and elective frozen embryo transfer (eFET) in comparison with fresh embryo transfer in assisted reproductive technology treatment among female and male patients before and after their first ART treatment cycle in a public health care setting? SUMMARY ANSWER: Despite concerns about the delay in embryo transfer associated with eFET, nearly 60% of the participants were in favor of eFET compared with fresh embryo transfer assuming that the clinical pregnancy rate was equivalent. WHAT IS KNOWN ALREADY: Vitrification and blastocyst transfer have considerably improved success rates after FET with ongoing pregnancy rates in frozen cycles approaching those seen in fresh treatment cycles. Furthermore, the risk of ovarian hyperstimulation syndrome (OHSS) is essentially eliminated in FET cycles, and FET may be beneficial to the endometrial and fetal development because a hormonal environment mirroring the natural cycle is enabled. However, the freeze-all strategy is not yet implemented as standard care. One reason is the presumption of negative patient attitudes towards a freeze-all embryo strategy. So far, no data regarding patients' attitudes on a freeze-all strategy have been published. STUDY DESIGN, SIZE, DURATION: This study was designed as a descriptive cross-sectional study including 165 fertility patients referred for their first ART treatment from December 2014 to June 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: All newly referred patients participating in a mandatory meeting before initiating ART treatment at the Fertility Clinic, Hvidovre Hospital, Copenhagen, Denmark were requested to fill in an online web-based questionnaire separately for men and women covering attitudes towards a freeze-all strategy, socio-demographic data and reproductive history. The patients were informed about both conventional fresh embryo transfer strategy and the freeze-all strategy prior to answering the questionnaire. MAIN RESULTS AND THE ROLE OF CHANCE: The total response rate was 77.1% (n = 165), and for women and men respectively 85.8 versus 66.0%. The female respondents were significantly more likely to consider the postponement of embryo transfer difficult compared to the male population (78.6 versus 35.5%; P < 0.001) and they were significantly more willing to accept a risk in treatment on own health to achieve a pregnancy than were the male respondents on their partners health (82.5 versus 96.8%; P = 0.025). However, 59.2% of the women and 59.7% of the men agreed that they would choose eFET over fresh embryo transfer if the chance of pregnancy were the same. Most of the patients furthermore agreed that the health of the mother and their baby was of highest importance. In the adjusted analyses we found no significant predictive factors for preferences towards a freeze-all strategy apart from a negative attitude towards delay of transfer in case of previous unsuccessful ART attempts. LIMITATIONS, REASONS FOR CAUTION: Selection bias cannot be excluded, as the total response rate was 77.1%. The hypothetical nature of the items may furthermore limit the validity of the results. In addition, the participants were from a single Fertility Clinic in the Capital Region of Denmark and may therefore not be representative for all fertility patients. WIDER IMPLICATIONS OF THE FINDINGS: In a clinical setting with similar pregnancy rates for eFET and fresh embryo transfer, these results indicate that patients, when given access to information on advantages and disadvantages of both fresh embryo transfer and eFET, are less prone to opt for fresh embryo transfer. This may be ground breaking for a patient-centered paradigm shift in routine ART treatment with a wider implementation of a freeze-all and eFET-strategy eliminating the risk of OHSS. STUDY FUNDING/COMPETING INTEREST(S): The Danish Council for Independent Research and Merck Serono supported the study. The study is part of the Reprounion Collaborative study, co-financed by the European Union, Interreg V ÖKS. No competing interests exist.

Prediction of the lower serum anti-Müllerian hormone threshold for ovarian stimulation prior to in-vitro fertilization using the Elecsys® AMH assay: a prospective observational study.

BACKGROUND: In assisted reproductive technology, prediction of treatment failure remains a great challenge. The development of more sensitive assays for measuring anti-Müllerian hormone (AMH) has allowed for the possibility to investigate if a lower threshold of AMH can be established predicting very limited or no response to maximal ovarian stimulation. METHODS: A prospective observational multicenter study of 107 women, < 40 years of age with regular menstrual cycle and serum AMH levels ≤ 12 pmol/L, treated with 300 IU/day of HP-hMG in a GnRH-antagonist protocol. AMH was measured before treatment start using the Elecsys® AMH assay by Roche Diagnostics. The ability of AMH to predict follicular development and ovarian response was assessed by receiver operating characteristics (ROC). Furthermore, the relationship between AMH at start of stimulation and cycle outcome was investigated using multivariate logistic regression analysis. RESULTS: Five out of 107 cycles (4.7%) were cancelled due to lack of follicular development and 60/107 (56%) women did not reach the classical hCG criteria for ovulation induction (≥ 3 follicles of ≥17 mm). An AMH threshold of 4 pmol/L predicted failure to reach the classical hCG criteria with 89% specificity and 53% sensitivity and an area under the curve (AUC) of 0.76 (95% CI 0.66-0.85). AMH predicted cycle cancellation due to lack of follicular development, using a cut-off value of 1.5 pmol/L, with a specificity of 96% and sensitivity of 80% (AUC = 0.92, 95% CI 0.79-1.00). A single-unit increase in AMH was associated with a 29% decrease in odds of failure to reach the classical hCG criteria (OR 0.71 95% CI 0.59-0.85, p < 0.01). The lowest AMH value compatible with a live birth was 1.3 pmol/L. CONCLUSIONS: Among women with a limited ovarian reserve, pre-treatment serum AMH levels significantly predicted failure to reach the classical hCG triggering criteria and predicted lack of follicular development using a new sensitive assay, but AMH was not suitable for withholding fertility treatment, as even very low levels were associated with live births. TRIAL REGISTRATION: Not relevant.

Concentration of soluble urokinase plasminogen activator receptor (suPAR) in the pre-ovulatory follicular fluid is associated with development of ovarian hyperstimulation syndrome during ovarian stimulation.

PURPOSE: Investigating whether pre-ovulatory follicular fluid (FF) levels of selected proteins differ between women who do or do not develop severe ovarian hyperstimulation syndrome (OHSS) and evaluate whether they potentially could guide a "freeze-all" strategy. METHODS: FF was collected during a randomized controlled trial comparing OHSS in antagonist versus agonist protocol including 1050 women in their first assisted reproductive technology (ART) cycle during year 2009-2013. The present sub-study is a matched case-control study comparing FF levels of soluble urokinase plasminogen activator receptor (suPAR), C-reactive protein, placental growth factor, vascular endothelial growth factor, and angiopoietins 1 and 2 in OHSS cases (n = 25, severe OHSS, and ≥ 15 oocytes), high-risk controls (n = 25, no OHSS, and ≥ 15 oocytes), and low-risk controls (n = 25, no OHSS, and 5-8 oocytes). RESULTS: FF level of suPAR differed significantly between the three groups (p = 0.018) with mean (SD) levels of 2.3 (0.4) µg/L, 2.6 (0.8) µg/L, and 2.8 (0.6) µg/L in OHSS cases, high-risk controls, and low-risk controls, respectively. Receiver operating characteristic curve analysis demonstrated that suPAR levels could predict severe OHSS (AUC 0.678; 95% CI 0.553-0.803) with a sensitivity of 64% and a specificity of 66%. None of the other investigated proteins differed between the three groups or between OHSS cases and combined controls. CONCLUSION: The pre-ovulatory FF level of suPAR was significantly lower in women developing severe OHSS, indicating that the plasminogen activator system could be involved in the pathophysiology of OHSS. However, suPAR did not provide a satisfying predictive value for the prediction of OHSS.

Cumulative live birth rates after one ART cycle including all subsequent frozen-thaw cycles in 1050 women: secondary outcome of an RCT comparing GnRH-antagonist and GnRH-agonist protocols.

STUDY QUESTION: Are cumulative live birth rates (CLBRs) similar in GnRH-antagonist and GnRH-agonist protocols for the first ART cycle including all subsequent frozen-thaw cycles from the same oocyte retrieval? SUMMARY ANSWER: The chances of at least one live birth following utilization of all fresh and frozen embryos after the first ART cycle are similar in GnRH-antagonist and GnRH-agonist protocols. WHAT IS KNOWN ALREADY: Reproductive outcomes of ART treatment are traditionally reported as pregnancies per cycle or per embryo transfer. However, the primary concern is the overall chance of a live birth. After the first ART cycle with fresh embryo transfer, we found live birth rates (LBRs) of 22.8% and 23.8% (P = 0.70) for the GnRH-antagonist and GnRH-agonist protocols, respectively. But with CLBRs including both fresh and frozen embryos from the first oocyte retrieval, chances of at least one live birth increases. There are no previous randomized controlled trials (RCTs) comparing CLBRs in GnRH-antagonist versus GnRH-agonist protocols. Previous studies on CLBR are either retrospective cohort studies including multiple fresh cycles or RCTs comparing single embryo transfer (SET) with double embryo transfer (DET). STUDY DESIGN, SIZE, DURATION: CLBR was a secondary outcome in a Phase IV, dual-center, open-label, RCT including 1050 women allocated to a short GnRH-antagonist or a long GnRH-agonist protocol in a 1:1 ratio over a 5-year period using a web-based concealed randomization code. The minimum follow-up time from the first IVF cycle was 2 years. The aim was to compare CLBR between the two groups following utilization of all fresh and frozen embryos from the first ART cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: All women referred for their first ART cycle at two public fertility clinics, <40 years of age were approached. A total of 1050 subjects were allocated to treatment and 1023 women started standardized ART protocols with recombinant human follitropin-ß (rFSH) stimulation. Day-2 SET was planned and additional embryos were frozen and used in subsequent frozen-thawed cycles. All pregnancies generated from oocyte retrieval during the first IVF cycle including fresh and frozen-thaw cycles were registered. Ongoing pregnancy was determined by ultrasonography at gestational week 7-9 and live birth was irrespective of the duration of gestation. CLBR was defined as at least one live birth per allocated woman after fresh and frozen cycles. Subjects were censored out after the first live birth. Cox proportional hazard model was used to evaluate the relative prognostic significance of female age, BMI, the number of retrieved oocytes and the diagnosis of infertility in relation to the CLBR. MAIN RESULTS AND THE ROLE OF CHANCE: Baseline characteristics were similar and equal proportions of patients continued with frozen-thaw (frozen embryo transfer, FET) cycles after their fresh ART cycle in the GnRH-antagonist and GnRH-agonist arms. When combining all fresh and frozen-thaw embryo transfers from first oocyte retrieval with a minimum of 2-year follow-up, the CLBR was 34.1% (182/534) in the GnRH-antagonist group versus 31.2% (161/516) in the GnRH-agonist group (odds ratio (OR):1.14; 95% CI: 0.88-1.48, P = 0.32). Mean time to the first live birth was 11.0 months in the GnRH-antagonist group compared to 11.5 months in the GnRH-agonist group (P < 0.01). The total number of deliveries from all FET cycles where embryos were thawed were higher in the antagonist group 64/330 (19.4%) compared to the agonist group 43/355 (12.1%) ((OR): 1.74; 95% CI: 1.14-2.66, P = 0.01). The evaluation of prognostic factors showed that more retrieved oocytes were associated with a significantly higher CLBR in both treatment groups. For the subgroup of obese women (BMI >30 kg/m2), the CLBR was significantly higher in the GnRH-antagonist group (P = 0.02). LIMITATIONS, REASONS FOR CAUTION: The duration of the trial is a possible limitation with introduction of new methods as 'Freeze all' and 'GnRH-agonist triggering', but as these treatments were used in only few women, a systematic bias is not likely. Blastocyst culture of surplus embryos for freezing was introduced to both groups simultaneously, thereby minimizing the risk of bias. Furthermore, with a minimum of 2-year follow-up, a minority (<1%) still had cryopreserved embryos and no live birth at the end of the trial. The post hoc prognostic covariate analyses with multiple strata should be interpreted with caution. Finally, the physicians were not blinded to GnRH treatment group after randomization. WIDER IMPLICATIONS OF THE FINDINGS: With the improvement of embryo culture, freezing and thawing methods as well as a strategy of elective SET, CLBR until first live birth provides an all-inclusive success rate for ART. When comparing GnRH-antagonist and GnRH-agonist protocols, we find similar CLBRs, despite more oocytes being retrieved in the GnRH-agonist protocol. STUDY FUNDING/COMPETING INTERESTS: An unrestricted research grant is funded by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (MSD). The funders had no influence on the data collection, analyses or conclusions of the study. No conflict of interests to declare. TRIAL REGISTRATION NUMBER: EudraCT #: 2008-005452-24. ClinicalTrial.gov: NCT00756028. TRIAL REGISTRATION DATE: 18 September 2008. DATE OF FIRST PATIENT'S ENROLLMENT: 14 January 2009.

Risk of severe ovarian hyperstimulation syndrome in GnRH antagonist versus GnRH agonist protocol: RCT including 1050 first IVF/ICSI cycles.

STUDY QUESTION: Is the risk of severe ovarian hyperstimulation syndrome (OHSS) similar in a short GnRH antagonist and long GnRH agonist protocol in first cycle IVF/ICSI patients less than 40 years of age?. SUMMARY ANSWER: There is an increased risk of severe OHSS in the long GnRH agonist group compared with the short GnRH antagonist protocol. WHAT IS KNOWN ALREADY?: In the most recent Cochrane review, the GnRH antagonist protocol was associated with a similar live birth rate (LBR), a similar on-going pregnancy rate (OPR), and a lower incidence of OHSS (odds ratio (OR) = 0.43 95% confidence interval (CI): 0.33-0.57) compared with the traditional GnRH agonist protocol. Previous trials comparing the two protocols mainly included selected patient populations, a limited number of patients and the applied OHSS criteria differed, making direct comparisons difficult. In two recent large meta-analyses, no significant differences in LBR (OR = 0.86; 95% CI: 0.72-1.02) or in the incidence of severe OHSS were reported, while others found a lower LBR (OR = 0.82; 95% CI: 0.68-0.97) and a reduced risk of severe OHSS using the GnRH antagonist protocol (OR = 0.60; 95% CI: 0.40-0.88). STUDY DESIGN, SIZE, DURATION: Phase IV, dual-centre, open-label, RCT including 1050 women allocated to either short GnRH antagonist or long GnRH agonist protocol in a 1:1 ratio and enrolled over a 5-year period using a web-based concealed randomization code. This is a superiority study designed to detect a difference in severe OHSS, the primary outcome, between the two groups with a power of 80% and stratified for age, assisted reproductive technology (ART) clinic and planned fertilization procedure (IVF/ICSI). The secondary aims were to compare rates of mild and moderate OHSS, positive plasma (p)-hCG, on-going pregnancy and live birth between the two arms. None of the women had undergone previous ART treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: All infertile women referred for their first IVF/ICSI at two public fertility clinics, less than 40 years of age and with no uterine malformations were asked to participate. A total of 1099 subjects were randomized, including women with poor ovarian reserve, polycystic ovary syndrome and irregular cycles. A total of 49 women withdrew their consent, thus 1050 subjects were allocated to the GnRH antagonist (n = 534) and agonist protocol (n = 516), respectively. In total 1023 women started recombinant human follitropin-ß (rFSH) stimulation, 528 in the GnRH antagonist group and 495 in the GnRH agonist group. All subjects were given a fixed rFSH dose of 150 IU or 225 IU according to age ≤36 years or >36 years, with the option to adjust dose at stimulation day 6. Clinical OHSS parameters were collected at oocyte retrieval, and Days 3 and 14 post-transfer. On-going pregnancy was determined by transvaginal ultrasonography at gestational weeks 7-9. In the intention-to-treat (ITT) analysis for reproductive outcomes, 1050 subjects were included. For the ITT analyses on OHSS 1023 subjects who started gonadotrophin stimulation were included. MAIN RESULTS AND THE ROLE OF CHANCE: The incidence of severe OHSS [5.1% (27/528) versus 8.9% (44/495) (difference in proportion percentage point (Δpp) = -3.8pp; 95% CI: -7.1 to -0.4; P = 0.02)] and moderate OHSS [10.2% (54/528) versus 15.6% (77/495) (Δpp = -5.3pp; 95% CI: -9.6 to -1.0; P = 0.01) ] was significantly lower in the GnRH antagonist group compared with the agonist group, respectively. In the GnRH antagonist and agonist group, respectively, 4.7% (25/528) versus 8.5% (42/495) women were seen by a physician due to OHSS (P = 0.01), and 1.7% (9/528) versus 3.6% (18/495) were admitted to hospital due to OHSS (P = 0.06). No women had ascites-puncture in the GnRH antagonist group versus 2.0% (10/495) in the GnRH agonist group (P < 0.01). LBRs were 22.8% (122/534) versus 23.8% (123/516) (Δpp = -1.0pp; 95% CI: -6.3 to 4.3; P = 0.70) and OPRs were 24.9% (133/528) versus 26.2% (135/516) (Δpp = -1.3pp; 95% CI: -6.7 to 4.2; P = 0.64) per randomized subject in the GnRH antagonist versus agonist group, with a mean number of 1.1 versus 1.2 embryos transferred in the two groups. Pregnancy rates (PR) per randomized subject, per started gonadotrophin stimulation and per embryo transfer were all similar in the two groups. LIMITATIONS, REASONS FOR CAUTION: A possible limitation is the duration of the trial, with new methods, such as 'freeze all' and 'GnRH agonist triggering', being developed during the trial, the new methods were sought avoided, however a total number of 32 women had 'freeze all' and 'GnRH agonist triggering' was performed in three cases. Ultrasonic measurements were performed by different physicians and inter-observer bias may be present. Measures of anti-Mullerian hormone and antral follicle count, to estimate ovarian reserve and thus predict risk of OHSS, were not performed. Finally, the physicians were not blinded to GnRH treatment group after randomization. WIDER IMPLICATIONS OF THE FINDINGS: The short GnRH antagonist protocol should be the protocol of choice for patients undergoing their first ART cycle in females <40 years of age including both low and high responders when an age-dependent initially fixed gonadotrophin dose is used, as an increased risk of severe OHSS and the associated complications is seen in the long GnRH agonist group and as PRs and LBRs are similar in the two groups. Patients at risk of OHSS particularly benefit from the short GnRH antagonist treatment as GnRH agonist triggering can be used. STUDY FUNDING/COMPETING INTERESTS: An unrestricted research grant is funded by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (MSD). The funders had no influence on the data collection, analyses or conclusions of the study. No conflict of interests to declare. TRIAL REGISTRATION NUMBER: EudraCT #: 2008-005452-24. ClinicalTrial.gov: NCT00756028. Trial registration date: 18 September 2008. Date of first patient's enrolment: 14 January 2009.