Circadian serum progesterone variations on the day of frozen embryo transfer in a modified natural cycle protocol.

STUDY QUESTION: Is there a circadian variation of serum progesterone (P) on the day of frozen embryo transfer (FET) in a modified natural cycle (mNC)? SUMMARY ANSWER: There is a statistically significant diurnal variation of serum P on the day of a FET in an mNC protocol. WHAT IS KNOWN ALREADY: In recent years, the proportion of FET cycles has increased dramatically. To further optimize pregnancy outcomes after FET, recent studies have focused on serum luteal P levels in both natural and artificially prepared FET cycles. Despite the different cut-off values proposed to define low serum P in the NC, it is generally accepted that lower serum P values (<10 ng/ml) around the day of FET are associated with negative reproductive outcomes. However, a single serum P measurement is not reliable given that P levels are prone to diurnal fluctuations and are impacted by patients' characteristics. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was conducted in a single university-affiliated fertility center, including 22 patients performing a single blastocyst mNC-FET from August 2022 to August 2023. Serum P levels were measured on the day of transfer at 08:00h, 12:00h, 16:00h, and 20:00h. Differences between P levels were compared using the Wilcoxon signed-rank test. The sample size was calculated to detect a difference of 15% between the first and last P measurements with a 5% false-positive rate and a 95% CI. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients with a normal BMI, between 18 and 40 years old, without uterine diseases were eligible. Patients utilizing donated oocytes were excluded. The mNC-FET protocol involved monitoring the normal ovarian cycle and triggering ovulation with an injection of 250 µg of choriogonadotropin alfa when a pre-ovulatory follicle (16-20 mm diameter) was visualized. The blastocyst was transferred seven days later. The patients were not supplemented with exogenous P at any time before the day of the FET. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age and BMI of the study population were 33.6 ± 3.8 years and 22.7 ± 1.8 kg/m2, respectively. Mean P values at 08:00h, 12:00h, 16:00h, and 20:00h were 14.6 ± 4.5, 14.7 ± 4.1, 12.9 ± 3.5, and 14.6 ± 4.3 ng/ml, respectively. The mean P levels at 16:00h were significantly lower compared to all other time points (P < 0.05: P = 0.007 between P at 8:00h and 16:00h; P = 0.003 between P at 12:00h and 16:00h; P = 0.007 between P at 16:00h and 20:00h). No statistically significant difference was observed between P values at the other time points (P > 0.05: P = 0.88 between P at 8:00h and 12:00h; P = 0.96 between P at 8:00h and 20:00h; P = 0.83 between P at 12:00h and 20:00h). LIMITATIONS, REASONS FOR CAUTION: The study's limitations include the small sample size that may cause a bias when the results are extrapolated to a larger subfertile population undergoing mNC-FET. Ideally, larger prospective trials including a more heterogeneous patient population would be necessary to validate our findings. WIDER IMPLICATIONS OF THE FINDINGS: The current study demonstrates the existence of a diurnal fluctuation of serum P on the day of mNC-FET highlighting the importance of a standardized time point for its measurement. This is especially important for considering clinical actions, such as additional exogenous P supplementation, when encountering P values lower than 10 ng/ml on the day of FET. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained for the study. The authors have no conflicts of interest to declare regarding the content of the study. TRIAL REGISTRATION NUMBER: NCT05511272.

Reproductive outcomes in women opting for fertility preservation after fertility-sparing surgery for borderline ovarian tumors.

PURPOSE: What are the reproductive outcomes of women who had fertility preservation (FP) using either oocyte or embryo vitrification after fertility-sparing surgery (FSS) for a borderline ovarian tumor (BOT)? METHODS: A retrospective, single-center cohort study was conducted between January 2013 and December 2021. Patients with BOT who resorted to FP by vitrifying oocytes or embryos were included. Both clinical and reproductive parameters were reviewed. The primary outcome was live birth. RESULTS: In total, thirteen patients who performed 31 FP cycles were included. Of those, six patients achieved eight live births after a mean follow-up period of 79 months. Three further pregnancies are still ongoing. All pregnancies/live births were obtained without using their cryopreserved oocytes or embryos. CONCLUSION: Women who had FSS for BOT have favorable prospects of live offspring, even without the need to use their cryopreserved material. Fertility preservation in patients with BOT has to be considered as a tool to mitigate the risk of infertility that may arise in case of BOT recurrence requiring castrating surgery.

Progestin primed ovarian stimulation using dydrogesterone from day 7 of the cycle onwards in oocyte donation cycles: a longitudinal study.

RESEARCH QUESTION: Does a progestin-primed ovarian stimulation (PPOS) protocol with dydrogesterone from cycle day 7 yield similar outcomes compared with a gonadotrophin-releasing hormone (GnRH) antagonist protocol in the same oocyte donors? DESIGN: This retrospective longitudinal study included 128 cycles from 64 oocyte donors. All oocyte donors had the same type of gonadotrophin and daily dose in both stimulation cycles. The primary outcome was the number of cumulus-oocyte complexes (COC) retrieved. RESULTS: The number of COC retrieved (mean ± SD 19.7 ± 10.8 versus 19.2 ± 8.3; P = 0.5) and the number of metaphase II oocytes (15.5 ± 8.4 versus 16.2 ± 7.0; P = 0.19) were similar for the PPOS and GnRH antagonist protocols, respectively. The duration of stimulation (10.5 ± 1.5 days versus 10.8 ± 1.5 days; P = 0.14) and consumption of gonadotrophins (2271.9 ± 429.7 IU versus 2321.5 ± 403.4 IU; P = 0.2) were also comparable, without any cases of premature ovulation. Nevertheless, there was a significant difference in the total cost of medication per cycle: €898.3 ± 169.9 for the PPOS protocol versus €1196.4 ± 207.5 (P < 0.001) for the GnRH antagonist protocol. CONCLUSION: The number of oocytes retrieved and number of metaphase II oocytes were comparable in both stimulation protocols, with the advantage of significant cost reduction in favour of the PPOS protocol compared with the GnRH antagonist protocol. No cases of premature ovulation were observed, even when progestin was started later in the stimulation.

Does dual oocyte retrieval with continuous FSH administration increase the number of mature oocytes in low responders? An open-label randomized controlled trial.

STUDY QUESTION: Is there an increase in the total number of metaphase II (MII) oocytes between a conventional ovarian stimulation (OS) and a double uninterrupted stimulation? SUMMARY ANSWER: There is no increase in the total number of MII oocytes when comparing one conventional OS to a continuous stimulation with double oocyte aspiration. WHAT IS KNOWN ALREADY: Based on the concept of multiple follicular waves, the combination of two stimulations in the same ovarian cycle has gained interest in patients with a low ovarian reserve. This so-called dual stimulation approach is usually characterized by a discontinuation of FSH administration for ∼5 days and appears to have a favourable impact on the number of retrieved oocytes without affecting the embryo quality or ploidy status. The outcomes of dual uninterrupted OS have not yet been studied. STUDY DESIGN, SIZE, DURATION: This was an open-label randomized controlled trial (RCT) with superiority design, performed in a single tertiary centre. Subjects were randomized with a 1:1 allocation into two groups between October 2019 and September 2021. All patients underwent a conventional stimulation with recombinant FSH. When two or more follicles of 17 mm were present, the final inclusion criterion was assessed; randomization occurred only in the presence of ≤9 follicles of ≥11 mm. In Group A, ovulation was triggered with hCG, and oocyte retrieval (OR) was performed 34-36 h later, followed by a fresh single or double embryo transfer (SET or DET) on Day 3/5. In Group B, ovulation was triggered with GnRH agonist, followed by another OS, without discontinuation of the FSH administration. In the presence of one or more follicles of ≥17 mm, the second stimulation was completed with hCG. A freeze-all strategy (Day 3/5) was applied for both retrievals, followed by transfer of one or two embryos in an artificially prepared frozen-thawed cycle. In the absence of one or more follicles of ≥17 mm after 13 additional days of stimulation, the second cycle was cancelled. All ORs were executed by a senior fertility specialist who was blinded for the first treatment, and all follicles >10 mm were aspirated, according to routine clinical practice. The primary outcome was the total number of MII oocytes. Patients were followed up until all embryos were transferred, or until live birth was achieved. Other secondary outcomes included the number of cumulus-oocyte complexes (COCs), the number of good quality embryos (Day 3/5), the ongoing pregnancy rate, and gonadotropin consumption. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients between 25 and 40 years old, with an anti-Müllerian hormone level of ≤1.5 ng/ml, antral follicle count of ≤6, or ≤5 oocytes after a previous stimulation, were included. At the start, 70 patients were eligible for participation in the trial, of whom 48 patients fulfilled the final inclusion criterium and were randomized. After drop-out of two patients, 23 patients were randomized to a single round of OS (Group A), and 23 patients were randomized to two uninterrupted rounds of OS (Group B). MAIN RESULTS AND THE ROLE OF CHANCE: Baseline characteristics were similar between both groups. The cumulative number of COCs and MII oocytes after completion of the second OR was similar in Group A and Group B [5.3 ± 2.7 versus 5.3 ± 3.0 (P = 0.95); 4.1 ± 2.4 versus 4.3 ± 2.7 (P = 0.77)]. Likewise, a comparable number of excellent and good quality embryos was available on Day 3 (3.0 ± 2.0 versus 2.7 ± 2.0; P = 0.63). In Group B, the cancellation rate due to insufficient response to the second round of stimulation was 39.1% (9/23). When focusing on the first stimulation in both groups, there were no significant differences regarding basal FSH, gonadotropin consumption, and the number of preovulatory follicles. After the first OR, the mean number of COC and MII oocytes was significantly higher in Group A (who had hCG triggering), compared to Group B (who had GnRH agonist triggering) [5.3 ± 2.7 versus 3.3 ± 2.2; difference 95% CI (0.54 to 3.45), P = 0.004 and 4.1 ± 2.4 versus 3.0 ± 2.2; difference 95% CI (-0.15 to 2.6), P = 0.05, respectively]. Likewise, the number of excellent and good quality embryos on Day 3 was significantly higher (3.0 ± 2.0 versus 1.9 ± 1.7; P = 0.02) in Group A. LIMITATIONS, REASONS FOR CAUTION: This study was powered to demonstrate superiority for the number of MII oocytes after dual stimulation. Investigating the impact of dual stimulation on pregnancy rates would have required a larger sample size. Furthermore, the heterogeneity in embryo vitrification and transfer policies precluded a correct comparison of embryologic outcomes between both groups. WIDER IMPLICATIONS OF THE FINDINGS: This is the first RCT investigating the role of continuous stimulation with double aspiration in low responders. Our results show no statistically significant differences in the cumulative number of MII oocytes between one conventional stimulation with fresh ET and two consecutive stimulations with a freeze-only approach. Furthermore, the observed suboptimal oocyte yield after agonist ovulation triggering in low responders in the dual uninterrupted OS group is a reason for concern and further scrutiny, given that previous RCTs have shown similar outcomes in normal and high responders after hCG and GnRH agonist triggers. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by a research grant from Organon. H.T. received honoraria for lectures and presentations from Abbott, Cooper Surgical, Gedeon-Richter, Cook, Goodlife, and Ferring. L.B. received fees for lectures from Merck & Organon and support for attending ESHRE 2023. M.D.V. reports fees for lectures from Ferring, Merck, Organon, IBSA, Gedeon Richter, and Cooper Surgical and support for attending ASRM 2023. S.M. received honoraria for lectures and presentations from Abbott, Cooper Surgical, Gedeon-Richter, IBSA, and Merck. C.B. was on the Advisory board and received consulting fees from Theramex and received honoraria for lectures and presentations from Abbott, Ferring, Gedeon-Richter, IBSA, and Merck. TRIAL REGISTRATION NUMBER: NCT03846544. TRIAL REGISTRATION DATE: 19 February 2019. DATE OF FIRST PATIENT'S ENROLMENT: 28 October 2019.

Oral dydrogesterone versus micronized vaginal progesterone for luteal phase support: a double-blind crossover study investigating pharmacokinetics and impact on the endometrium.

STUDY QUESTION: How do plasma progesterone (P) and dydrogesterone (D) concentrations together with endometrial histology, transcriptomic signatures, and immune cell composition differ when oral dydrogesterone (O-DYD) or micronized vaginal progesterone (MVP) is used for luteal phase support (LPS)? SUMMARY ANSWER: Although after O-DYD intake, even at steady-state, plasma D and 20αdihydrodydrogesterone (DHD) concentrations spiked in comparison to P concentrations, a similar endometrial signature was observed by histological and transcriptomic analysis of the endometrium. WHAT IS KNOWN ALREADY: O-DYD for LPS has been proven to be noninferior compared to MVP in two phase III randomized controlled trials. Additionally, a combined individual participant data and aggregate data meta-analysis indicated that a higher pregnancy rate and live birth rate may be obtained in women receiving O-DYD versus MVP for LPS in fresh IVF/ICSI cycles. Little data are available on the pharmacokinetic (PK) profiles of O-DYD versus MVP and their potential molecular differences at the level of the reproductive organs, particularly at the endometrial level. STUDY DESIGN, SIZE, DURATION: Thirty oocyte donors were planned to undergo two ovarian stimulation (OS) cycles with dual triggering (1.000 IU hCG + 0.2 mg triptorelin), each followed by 1 week of LPS: O-DYD or MVP, in a randomized, cross-over, double-blind, double-dummy fashion. On both the first and eighth days of LPS, serial blood samples upon first dosing were harvested for plasma D, DHD, and P concentration analyses. On Day 8 of LPS, an endometrial biopsy was collected for histologic examination, transcriptomics, and immune cell analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: All oocyte donors were <35 years old, had regular menstrual cycles, no intrauterine contraceptive device, anti-Müllerian hormone within normal range and a BMI ≤29 kg/m2. OS was performed on a GnRH antagonist protocol followed by dual triggering (1.000 IU hCG + 0.2 mg triptorelin) as soon as ≥3 follicles of 20 mm were present. Following oocyte retrieval, subjects initiated LPS consisting of MVP 200 mg or O-DYD 10 mg, both three times daily. D, DHD, and P plasma levels were measured using liquid chromatography-tandem mass spectrometry. Histological assessment was carried out using the Noyes criteria. Endometrial RNA-sequencing was performed for individual biopsies and differential gene expression was analyzed. Endometrial single-cell suspensions were created followed by flow cytometry for immune cell typing. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 21 women completed the entire study protocol. Subjects and stimulation characteristics were found to be similar between groups. Following the first dose of O-DYD, the average observed maximal plasma concentrations (Cmax) for D and DHD were 2.9 and 77 ng/ml, respectively. The Cmax for D and DHD was reached after 1.5 and 1.6 h (=Tmax), respectively. On the eighth day of LPS, the first administration of that day gave rise to a Cmax of 3.6 and 88 ng/ml for D and DHD, respectively. For both, the observed Tmax was 1.5 h. Following the first dose of MVP, the Cmax for P was 16 ng/ml with a Tmax of 4.2 h. On the eighth day of LPS, the first administration of that day showed a Cmax for P of 21 ng/ml with a Tmax of 7.3 h. All 42 biopsies showed endometrium in the secretory phase. The mean cycle day was 23.9 (±1.2) in the O-DYD group versus 24.0 (±1.3) in the MVP group. RNA-sequencing did not reveal significantly differentially expressed genes between samples of both study groups. The average Euclidean distance between samples following O-DYD was significantly lower than following MVP (respectively 12.1 versus 18.8, Mann-Whitney P = 6.98e-14). Immune cell profiling showed a decrease of CD3 T-cell, γδ T-cell, and B-cell frequencies after MVP treatment compared to O-DYD, while the frequency of natural killer (NK) cells was significantly increased. LIMITATIONS, REASONS FOR CAUTION: The main reason for caution is the small sample size, given the basic research nature of the project. The plasma concentrations are best estimates as this was not a formal PK study. Whole tissue bulk RNA-sequencing has been performed not correcting for bias caused by different tissue compositions across biopsies. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study comparing O-DYD/MVP, head-to-head, in a randomized design on a molecular level in IVF/ICSI. Plasma serum concentrations suggest that administration frequency is important, in addition to dose, specifically for O-DYD showing a rapid clearance. The molecular endometrial data are overall comparable and thus support the previously reported noninferior reproductive outcomes for O-DYD as compared to MVP. Further research is needed to explore the smaller intersample distance following O-DYD and the subtle changes detected in endometrial immune cells. STUDY FUNDING/COMPETING INTEREST(S): Not related to this work, C.Bl. has received honoraria for lectures, presentations, manuscript writing, educational events, or scientific advice from Abbott, Ferring, Organon, Cooper Surgical, Gedeon-Richter, IBSA, and Merck. H.T. has received honoraria for lectures, presentations, manuscript writing, educational events, or scientific advice from Abbott, Ferring, Cooper Surgical, Gedeon-Richter, Cook, and Goodlife. S.M. has received honoraria for lectures, presentations, educational events, or scientific advice from Abbott, Cooper Surgical, Gedeon-Richter, IBSA, and Merck and Oxolife. G.G. has received honoraria for lectures, presentations, educational events, or scientific advice from Merck, MSD, Organon, Ferring, Theramex, Gedeon-Richter, Abbott, Biosilu, ReprodWissen, Obseva, PregLem, Guerbet, Cooper, Igyxos, and OxoLife. S.V.-S. is listed as inventor on two patents (WO2019115755A1 and WO2022073973A1), which are not related to this work. TRIAL REGISTRATION NUMBER: EUDRACT 2018-000105-23.

A 10-year follow-up of reproductive outcomes in women attempting motherhood after elective oocyte cryopreservation.

STUDY QUESTION: Which reproductive treatment outcomes are observed in women who underwent elective oocyte cryopreservation (EOC) and who returned to the clinic with a desire for a child? SUMMARY ANSWER: Whether to warm oocytes or to first use fresh own oocytes for ART depends on age upon returning, but both strategies result in favorable reproductive outcomes. WHAT IS KNOWN ALREADY: Most affluent countries have observed a trend toward postponement of childbearing, and EOC is increasingly used based on the assumption that oocytes cryopreserved at a younger age may extend a woman's reproductive lifespan and mitigate her age-related fertility decline. Although most follow-up studies after EOC have focused on women who requested oocyte warming, a substantial proportion of women who do not conceive naturally will embark on fertility treatment without using their cryopreserved oocytes. Reports on reproductive outcomes in past EOC users are scarce, and the lack of reproductive treatment algorithms in this group of women hampers counseling toward the most efficient clinical strategy. STUDY DESIGN, SIZE, DURATION: This retrospective observational single-center study encompasses 843 women who had elective oocyte vitrification between 2009 and 2019 at our fertility clinic. Women who underwent fertility preservation for medical or oncological reasons were excluded. This study describes the outcomes of the diverse reproductive treatment strategies performed until May 2022 in women returning to our clinic to attempt motherhood. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using descriptive statistics, patient characteristics and data of ovarian stimulation (OS) of EOC cycles were analyzed, as well as data related to OS and laboratory data of ART in women who pursued fertility treatment with and/or without using their cryopreserved oocytes. The primary outcome was live birth rate (LBR) per patient after oocyte warming and after ART using fresh oocytes. Secondary outcomes were return rate, utilization rate of the cryopreserved oocytes, laboratory outcomes upon return, and LBR per embryo transfer. A multivariable regression model was developed to identify factors associated with the decision to thaw oocytes as the primary strategy and factors associated with ongoing pregnancy upon return to the clinic. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1353 EOC cycles (mean ± SD, 1.6 ± 0.9 per patient) were performed. At the time of EOC, the mean age was 36.5 ± 2.8 years, mean anti-Müllerian hormone (AMH) was 2.3 ± 2.0 ng/ml, and 174 (20.6%) women had a partner. On average, 13.9 ± 9.2 mature oocytes were cryopreserved. Two hundred thirty-one (27.4%) women returned to the clinic, an average of 39.9 ± 23.4 months after EOC. Upon returning, their mean age was 40.4 ± 3.1 years, mean AMH was 1.5 ± 1.5 ng/ml, and 158/231 (68.3%) patients had a partner. As a primary approach, 110/231 (47.6%) past EOC users embarked on oocyte warming, 50/231 (21.6%) had intrauterine insemination, and 71/231 (30.7%) had ART using fresh own oocytes. Cumulative LBR (CLBR) was 45.9% (106/231) notwithstanding a miscarriage rate (MR) of 30.7% (51/166) in the entire cohort. In total, 141 women performed oocyte warming at some stage in their treatment trajectory. A subset of 90/231 (39.0%) patients exclusively had oocyte warming (41.6 ± 3.0 years, with 10.0 ± 5.2 oocytes warmed per patient). 52/231 (22.5%) patients exclusively had ART using fresh own oocytes (mean age of 39.0 ± 2.8 years, with 9.9 ± 7.4 mature oocytes retrieved per patient). CLBR was 37/90 (41.1%) in the oocyte warming-only group and 25/52 (48.1%) in the OS-only group. MR/transfer was 25.0% and 29.3% in the oocyte warming-only group and the OS-only group, respectively. LIMITATIONS, REASONS FOR CAUTION: Both sample size and the retrospective design are limitations of this study. The decision to embark on a specific reproductive treatment strategy was based on patient preference, after counseling on their treatment options. This precludes direct comparison of the efficiency of reproductive treatment options in past EOC users in this study. WIDER IMPLICATIONS OF THE FINDINGS: Reporting on clinical outcomes of women who underwent EOC and returned to the clinic to embark on divergent reproductive treatment strategies is mandatory to establish guidelines for best clinical practice in this growing patient population. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Enzymatic tissue processing after testicular biopsy in non-obstructive azoospermia enhances sperm retrieval.

STUDY QUESTION: What is the added value of enzymatic processing of testicular biopsies on testicular sperm retrieval (SR) rates for patients with non-obstructive azoospermia (NOA)? SUMMARY ANSWER: In addition to mechanical mincing, enzymatic digestion increased SR rates in testicular biopsies of NOA patients. WHAT IS KNOWN ALREADY: Many studies focus on the surgical approach to optimize recovery of testicular sperm in NOA, and in spite of that, controversy still exists about whether the type of surgery makes any difference as long as multiple biopsies are taken. Few studies, however, focus on the role of the IVF laboratory and the benefit of additional lab procedures, e.g. enzymatic digestion, in order to optimize SR rates. STUDY DESIGN SIZE DURATION: This retrospective single-center cohort study included all patients who underwent their first testicular sperm extraction (TESE) by open multiple-biopsy method between January 2004 and July 2022. Only patients with a normal karyotype, absence of Y-q deletions and a diagnosis of NOA based on histology were included. The primary outcome was SR rate after mincing and/or enzymes. The secondary outcome was cumulative live birth (CLB) after ICSI with fresh TESE and subsequent ICSI cycles with frozen TESE. PARTICIPANTS/MATERIALS SETTING METHODS: Multiple biopsies were obtained from the testis, unilaterally or bilaterally, on the day of oocyte retrieval. Upon mechanical mincing, biopsies were investigated for 30 min; when no or insufficient numbers of spermatozoa were observed, enzymatic treatment was performed using Collagenase type IV. Multivariable regression analysis was performed to predict CLB per TESE by adjusting for the following confounding factors: male FSH level, female age, and requirement of enzymatic digestion to find sperm. MAIN RESULTS AND THE ROLE OF CHANCE: We included 118 patients, of whom 72 (61.0%) had successful SR eventually. Spermatozoa were retrieved after mechanical mincing for 28 patients (23.7%; 28/118) or after additional enzymatic digestion for another 44 patients (37.2%; 44/118). Thus, of the 90 patients requiring enzymatic digestion, sperm were retrieved for 44 (48.9%). Male characteristics were not different between patients with SR after mincing or enzymatic digestion, in regard to mean age (34.5 vs 34.5 years), testis volume (10.2 vs 10.6 ml), FSH (17.8 vs 16.9 IU/l), cryptorchidism (21.4 vs 34.1%), varicocele (3.6 vs 4.6%), or histological diagnosis (Sertoli-cell only 53.6 vs 47.7%, maturation arrest 21.4 vs 38.6%, sclerosis/atrophy 25.0 vs 13.6%).Of the 72 patients with sperm available for ICSI, 23/72 (31.9%) achieved a live birth (LB) after the injection with fresh testicular sperm (and fresh or frozen embryo transfers). Of the remaining 49 patients without LB, 34 (69.4%) had supernumerary testicular sperm frozen. Of these 34 patients, 19 (55.9%) continued ICSI with frozen testicular sperm, and 9/19 (47.4%) had achieved an LB after ICSI with frozen testicular sperm. Thus, the total CLB was 32/118 (27.1%) per TESE or 32/72 (44.4%) per TESE with sperm retrieved.Of the female characteristics (couples with sperm available), only female age (30.3 vs 32.7 years; P = 0.042) was significantly lower in the group with a LB, compared to those without.The CLB with testicular sperm obtained after enzymatic digestion was 31.8% (14/44), while the CLB with sperm obtained after mincing alone was 64.3% (18/28). Multivariable logistic regression analysis showed that when enzymatic digestion was required, it was associated with a significant decrease in CLB per TESE (OR: 0.23 (0.08-0.7); P = 0.01). LIMITATIONS REASONS FOR CAUTION: Limitations of the study are related to the retrospective design. However, the selection of only patients with NOA, and specific characteristics (normal karyotype and absence Y-q deletion) and having their first TESE, strengthens our findings. WIDER IMPLICATIONS OF THE FINDINGS: Enzymatic processing increases the SR rate from testicular biopsies of NOA patients compared to mechanical mincing only, demonstrating the importance of an appropriate laboratory protocol. However, NOA patients should be counseled that when sperm have been found after enzymatic digestion, their chances to father a genetically own child may be lower compared to those not requiring enzymatic digestion. STUDY FUNDING/COMPETING INTERESTS: None reported. TRIAL REGISTRATION NUMBER: N/A.

Spermatogenesis after gonadotoxic childhood treatment: follow-up of 12 patients.

STUDY QUESTION: What is the long-term impact of presumed gonadotoxic treatment during childhood on the patient's testicular function at adulthood? SUMMARY ANSWER: Although most patients showed low testicular volumes and some degree of reproductive hormone disruption 12.3 (2.3-21.0) years after gonadotoxic childhood therapy, active spermatogenesis was demonstrated in the semen sample of 8 out of the 12 patients. WHAT IS KNOWN ALREADY: In recent decades, experimental testicular tissue banking programmes have been set up to safeguard the future fertility of young boys requiring chemo- and/or radiotherapy with significant gonadotoxicity. Although the risk of azoospermia following such therapies is estimated to be high, only limited long-term data are available on the reproductive potential at adulthood. STUDY DESIGN SIZE DURATION: This single-centre prospective cohort study was conducted between September 2020 and February 2023 and involved 12 adult patients. PARTICIPANTS/MATERIALS SETTING METHODS: This study was carried out in a tertiary care centre and included 12 young adults (18.1-28.3 years old) who had been offered testicular tissue banking prior to gonadotoxic treatment during childhood. All patients had a consultation and physical examination with a fertility specialist, a scrotal ultrasound to measure the testicular volumes and evaluate the testicular parenchyma, a blood test for assessment of reproductive hormones, and a semen analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Testicular tissue was banked prior to the gonadotoxic treatment for 10 out of the 12 included patients. Testicular volumes were low for 9 patients, and 10 patients showed some degree of reproductive hormone disruption. Remarkably, ongoing spermatogenesis was demonstrated in 8 patients at a median 12.3 (range 2.3-21.0) years post-treatment. LIMITATIONS REASONS FOR CAUTION: This study had a limited sample size, making additional research with a larger study population necessary to verify these preliminary findings. WIDER IMPLICATIONS OF THE FINDINGS: These findings highlight the need for multicentric research with a larger study population to establish universal inclusion criteria for immature testicular tissue banking. STUDY FUNDING/COMPETING INTERESTS: This study was conducted with financial support from the Research Programme of the Research Foundation-Flanders (G010918N), Kom Op Tegen Kanker, and Scientific Fund Willy Gepts (WFWG19-03). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: NCT04202094; https://clinicaltrials.gov/ct2/show/NCT04202094?id=NCT04202094&draw=2&rank=1 This study was registered on 6 December 2019, and the first patient was enrolled on 8 September 2020.

The effect of polymorphisms in FSHR gene on late follicular phase progesterone and estradiol serum levels in predicted normoresponders.

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 <38 years old undergoing their first or second OS cycle. The prevalence of late follicular phase progesterone elevation (PE), as well as mean serum progesterone and estradiol levels on the day of trigger were compared between the different FSHR SNPs genotypes. PE was defined as >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.

Does the dose or type of gonadotropins affect the reproductive outcomes of poor responders undergoing modified natural cycle IVF (MNC-IVF)?

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 < 75 IU/d [11/1398 (0.8 %)] or 75 to < 100 IU/d [1303/1398 (93.2 %)] or ≥ 100 to 150 IU/d [84/1398 (6 %)]. Patients were stimulated with rFSH [251/1398 (18 %)], uFSH [45/1398 (3.2 %)] or hp-hMG [1102/1398 (78.8 %)]. Clinical pregnancy rate was 119/1398 (8.5 %). Live birth was achieved in 80/1398 (5.7 %) of cycles. There was no significant difference in rates of pregnancy and live birth across different types and doses of gonadotropins. The GEE multivariate regression analysis, adjusting for relevant confounders, showed that the type of treatment strategy (rFSH/uFSH/hp-hMG) and the daily dose of gonadotropins were not associated with live birth rates (LBR) (p value 0.08 and 0.8, respectively). CONCLUSIONS: The type and daily dose of gonadotropins do not affect the reproductive outcome of poor responders undergoing MNC-IVF.

Impact of cell loss after warming of human vitrified day 3 embryos on obstetric outcome in single frozen embryo transfers.

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.

First live birth after fertility preservation using vitrification of oocytes in a woman with mosaic Turner syndrome.

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.

Is a freeze-all policy the optimal solution to circumvent the effect of late follicular elevated progesterone? A multicentric matched-control retrospective study analysing cumulative live birth rate in 942 non-elective freeze-all cycles.

STUDY QUESTION: Is late follicular elevated progesterone (LFEP) in the fresh cycle hindering cumulative live birth rates (CLBRs) when a freeze only strategy is applied? SUMMARY ANSWER: LFEP in the fresh cycle does not affect the CLBR of the frozen transfers in a freeze only approach, nor the embryo freezing rate. WHAT IS KNOWN ALREADY: Ovarian stimulation promotes the production of progesterone (P) which has been demonstrated to have a deleterious effect on IVF outcomes. While there is robust evidence that this elevation produces impaired endometrial receptivity, the impact on embryo quality remains a matter of debate. In particular, previous studies have shown that LFEP is associated with a hindered CLBR. However, most clinical insight on the effect of progesterone on embryo quality in terms of CLBRs have focused on embryo transfers performed after the fresh transfer, thus excluding the first embryo of the cohort. To be really informative on the possible detrimental effects of LFEP, evidence should be derived from freeze-all cycles where no fresh embryo transfer is performed in the presence of progesterone elevation, and the entire cohort of embryos is cryopreserved. STUDY DESIGN, SIZE, DURATION: This was a matched case-control, multicentre (three centres), retrospective analysis including all GnRH antagonist ICSI cycles in which a freeze all (FA) policy of embryos on day 3/5/6 of embryonic development was applied between 2012 and 2018. A total of 942 patients (471 cases with elevated P and 471 matched controls with normal P values) were included in the analysis. Each patient was included only once. PARTICIPANTS/MATERIALS, SETTING, METHODS: The sample was divided according to the following P levels on the day of ovulation triggering: <1.50 ng/ml and ≥1.50 ng/ml. The matching of the controls was performed according to age (±1 year) and number of oocytes retrieved (±10%). The main outcome was CLBR defined as a live-born delivery after 24 weeks of gestation. MAIN RESULTS AND THE ROLE OF CHANCE: The baseline characteristics of the two groups were similar. Estradiol levels on the day of trigger were significantly higher in the elevated P group. There was no significant difference in terms of fertilisation rate between the two groups. The elevated P group had significantly more cleavage stage frozen embryos compared to the normal P group while the total number of cryopreserved blastocyst stage embryos was the same. The CLBR did not differ between the two study groups (29.3% and 28.2% in the normal versus LFEP respectively, P = 0.773), also following confounder adjustment using multivariable GEE regression analysis (accounting for age at oocyte retrieval, total dose of FSH, progesterone levels on the day of ovulation trigger, day of freezing, at least one top-quality embryo transferred and number of previous IVF cycles, as the independent variables). LIMITATIONS, REASONS FOR CAUTION: This is a multicentre observational study based on a retrospective data analysis. Better extrapolation of the results could be validated by performing a prospective analysis. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study demonstrating that LFEP in the fresh cycle does not hinder CLBR of the subsequent frozen cycles in a FA approach. Thus, a FA strategy circumvents the issue of elevated P in the late follicular phase. STUDY FUNDING/COMPETING INTEREST(S): No funding was received for this study. Throughout the study period and manuscript preparation, authors were supported by departmental funds from: Centre for Reproductive Medicine, Brussels, Belgium; Infertility Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Centro Scienze Natalità, San Raffaele Scientific Institute, Milan, Italy; and IVI-RMA, Lisbon, Portugal. E.S. has competing interests with Ferring, Merck-Serono, Theramex and Gedeon-Richter outside the submitted work. E.P. reports grants from Ferring, grants and personal fees from Merck-Serono, grants and personal fees from MSD and grants from IBSA outside the submitted work. All the other authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Parameters of poor prognosis in preimplantation genetic testing for monogenic disorders.

STUDY QUESTION: What is the likelihood of success of a single cycle of preimplantation genetic testing for monogenic disorders (PGT-M), measured as the cumulative live birth rate (CLBR) and based on various patient demographics? SUMMARY ANSWER: For all women aged ≤40 years, the CLBR was at least 10% when the number of oocytes was ≥7 (range 10-30%) or was at least 5% when the number of oocytes was ≥3 (range 5-17%). WHAT IS KNOWN ALREADY: The number of oocytes is significantly associated with the number of embryos for genetic testing and the clinical outcome in PGT-M. Embryos diagnosed as affected or embryos that remain without diagnosis cannot be used for embryo transfer. The size of the group of embryos non-suitable for transfer varies between 25% and 81%, depending on the indication. Thus, PGT-M is more likely to be more severely impacted by suboptimal ovarian response, poor fertilization and suboptimal embryo development than conventional IVF/ICSI schemes without PGT. STUDY DESIGN, SIZE, DURATION: This was a single-centre retrospective comparative cohort study, of cycles between January 2011 and December 2015. A total number of 2265 PGT-M cycles were compared to 2833 conventional ICSI cycles. The principal aim of our study was the identification of the parameters of poor CLBR in couples undergoing PGT-M using multiplex short tandem repeat (STR) markers on blastomere biopsy DNA. The secondary aim was to compare the parameters of poor CLBR of the PGT-M population to those of couples undergoing ICSI without PGT. PARTICIPANTS/MATERIALS, SETTING, METHODS: The baseline characteristics of the PGT-M group were compared to the conventional ICSI group. A multiple regression analysis was applied to account for the following potential confounding factors: female age, number of previous ART cycles, number of oocytes/suitable embryos for transfer and dosage of gonadotrophins used for ovarian stimulation. MAIN RESULTS AND THE ROLE OF CHANCE: The PGT-M group was younger (female age 32.0 vs 34.5 years), had a higher number of previous ART cycles (1.1 vs 0.9 cycles) and used more gonadotrophins (2367 vs 1984 IU). Per cycle, the PGT-M group had more retrieved oocytes (11.8 vs 8.3 oocytes), fewer suitable embryos for transfer (1.7 vs 2.8 embryos) and a lower CLBR (29.4% vs 35.0%). Multiple regression analysis showed that the CLBR in the PGT-M group was significantly influenced by female age, the number of previous ART cycles, the number of oocytes and the dose of ovarian stimulation. In both groups, the predicted CLBR increased with increasing numbers of oocytes and suitable embryos. At least two retrieved oocytes or one embryo per single PGT-M cycle could confer an estimated CLBR above 10%. By assessing female age and the number of retrieved oocytes together, it was shown that for all women aged ≤40 years, the predicted CLBR per single PGT-M cycle was ≥10% when the number of oocytes was ≥7 or was ≥5% when the number of oocytes was ≥3. LIMITATIONS, REASONS FOR CAUTION: Despite the large sample size, the findings are confined by limited confounder adjustment and the lack of specific PGT-M comparators. WIDER IMPLICATIONS OF THE FINDINGS: This study aimed to describe the likelihood of success of PGT-M treatment, measured as CLBR, based on various patient demographics. In a PGT-M program, couples need to be informed of the prognosis more specifically when it is futile. The table of predicted CLBRs presented in this study is a useful tool in counselling PGT-M couples for making reproductive choices. STUDY FUNDING/COMPETING INTEREST(S): No funding was required and there are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

The effect of polymorphisms in FSHR and FSHB genes on ovarian response: a prospective multicenter multinational study in Europe and Asia.

STUDY QUESTION: Does the presence of single nucleotide polymorphisms (SNPs) in the FSH receptor gene (FSHR) and/or FSH beta subunit-encoding gene (FSHB) influence ovarian response in predicted normal responders treated with rFSH? SUMMARY ANSWER: The presence of FSHR SNPs (rs6165, rs6166, rs1394205) has a statistically significant impact in ovarian response, although this effect is of minimal clinical relevance in predicted normal responders treated with a fixed dose of 150 IU rFSH. WHAT IS KNOWN ALREADY: Ovarian reserve markers have been a breakthrough in response prediction following ovarian stimulation. However, a significant percentage of patients show a disproportionate lower ovarian response, as compared with their actual ovarian reserve. Studies on pharmacogenetics have demonstrated a relationship between FSHR or FSHB genotyping and drug response, suggesting a potential effect of individual genetic variability on ovarian stimulation. However, evidence from these studies is inconsistent, due to the inclusion of patients with variable ovarian reserve, use of different starting gonadotropin doses, and allowance for dose adjustments during treatment. This highlights the necessity of a well-controlled prospective study in a homogenous population treated with the same fixed protocol. STUDY DESIGN, SIZE, DURATION: We conducted a multicenter multinational prospective study, including 368 patients from Vietnam, Belgium, and Spain (168 from Europe and 200 from Asia), from November 2016 until June 2019. All patients underwent ovarian stimulation followed by oocyte retrieval in an antagonist protocol with a fixed daily dose of 150 IU rFSH until triggering. Blood sampling and DNA extraction was performed prior to oocyte retrieval, followed by genotyping of four SNPs from FSHR (rs6165, rs6166, rs1394205) and FSHB (rs10835638). PARTICIPANTS/MATERIALS, SETTING, METHODS: Eligible were predicted normal responder women <38 years old undergoing their first or second ovarian stimulation cycle. Laboratory staff and clinicians were blinded to the clinical results and genotyping, respectively. The prevalence of hypo-responders, the number of oocytes retrieved, the follicular output rate (FORT), and the follicle to oocyte index (FOI) were compared between different FSHR and FSHB SNPs genotypes. MAIN RESULTS AND THE ROLE OF CHANCE: The prevalence of derived allele homozygous SNPs in the FSHR was rs6166 (genotype G/G) 15.8%, rs6165 (genotype G/G) 34.8%, and rs1394205 (genotype A/A) 14.1%, with significant differences between Caucasian and Asian women (P < 0.001). FSHB variant rs10835638 (c.-211 G>T) was very rare (0.5%). Genetic model analysis revealed that the presence of the G allele in FSHR variant rs6166 resulted in less oocytes retrieved when compared to the AA genotype (13.54 ± 0.46 vs 14.81 ± 0.61, estimated mean difference (EMD) -1.47 (95% CI -2.82 to -0.11)). In FSHR variant rs1394205, a significantly lower number of oocytes was retrieved in patients with an A allele when compared to G/G (13.33 ± 0.41 vs 15.06 ± 0.68, EMD -1.69 (95% CI -3.06 to -0.31)). A significantly higher prevalence of hypo-responders was found in patients with the genotype A/G for FSHR variant rs6166 (55.9%, n = 57) when compared to A/A (28.4%, n = 29), ORadj 1.87 (95% CI 1.08-3.24). No significant differences were found regarding the FORT across the genotypes for FSHR variants rs6166, rs6165, or rs1394205. Regarding the FOI, the presence of the G allele for FSHR variant rs6166 resulted in a lower FOI when compared to the A/A genotype, EMD -13.47 (95% CI -22.69 to -4.24). Regarding FSHR variant rs6165, a lower FOI was reported for genotype A/G (79.75 ± 3.35) when compared to genotype A/A (92.08 ± 6.23), EMD -13.81 (95% CI -25.41 to -2.21). LIMITATIONS, REASONS FOR CAUTION: The study was performed in relatively young women with normal ovarian reserve to eliminate biases related to age-related fertility decline; thus, caution is needed when extrapolating results to older populations. In addition, no analysis was performed for FSHB variant rs10835638 due to the very low prevalence of the genotype T/T (n = 2). WIDER IMPLICATIONS OF THE FINDINGS: Based on our results, genotyping FSHR SNPs rs6165, rs6166, rs1394205, and FSHB SNP rs10835638 prior to initiating an ovarian stimulation with rFSH in predicted normal responders should not be recommended, taking into account the minimal clinical impact of such information in this population. Future research may focus on other populations and other genes related to folliculogenesis or steroidogenesis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by an unrestricted grant by Merck Sharp & Dohme (MSD). N.P.P. reports grants and/or personal fees from MSD, Merck Serono, Roche Diagnostics, Ferring International, Besins Healthcare, Gedeon Richter, Theramex, and Institut Biochimique SA (IBSA). N.L.V. and M.T.H. report consultancy and conference fees from Merck, Ferring, and MSD, outside the submitted work. P.D. has received honoraria for lecturing and/or research grants from MSD, Ferring International, and Merck. D.S. reports grants and/or personal fees from MSD, Ferring International, Merck Serono, Cook, and Gedeon Richter. A.R.N., B.A.M., C.S., J.M., L.H.L., P.Q.M.M., H.T., and S.G. report no conflict of interests. TRIAL REGISTRATION NUMBER: NCT03007043.

Effect of the oxytocin receptor antagonist nolasiban on pregnancy rates in women undergoing embryo transfer following IVF: analysis of three randomised clinical trials.

STUDY QUESTION: Does a single oral dose of nolasiban 900 mg administered 4 h before embryo transfer (ET) increase pregnancy rates in women undergoing IVF? SUMMARY ANSWER: In an individual patient data (IPD) meta-analysis of three clinical trials, a single oral dose of nolasiban 900 mg was associated with an increased ongoing pregnancy rate of an absolute 5% (relative 15%). WHAT IS KNOWN ALREADY: Several clinical studies have shown that blocking activation of oxytocin receptors by an oxytocin receptor (OTR) antagonist has the potential to decrease uterine contractions, increase endometrial perfusion and enhance endometrial decidualisation and other parameters of endometrial receptivity. It has been hypothesised that antagonism of oxytocin receptors could improve the likelihood of successful embryo implantation and thus increase pregnancy and live birth rates following ET. STUDY DESIGN, SIZE, DURATION: This is an analysis of three randomised, double-blind, placebo-controlled trials, which randomised 1836 subjects between 2015 and 2019. We describe the results of a meta-analysis of individual participant data (IPD) from all three trials and the pre-specified analyses of each individual trial. PARTICIPANT/MATERIAL, SETTING, METHODS: Participants were patients undergoing ET following IVF/ICSI in 60 fertility centres in 11 European countries. Study subjects were below 38 years old and had no more than one previously failed cycle. They were randomised to a single oral dose of nolasiban 900 mg (n = 846) or placebo (n = 864). In IMPLANT 1, additional participants were also randomised to nolasiban 100 mg (n = 62) or 300 mg (n = 60). Fresh ET of one good quality embryo (except in IMPLANT 1 where transfer of two embryos was allowed) was performed on Day 3 or Day 5 after oocyte retrieval, approximately 4 h after receiving the study treatment. Serum hCG levels were collected at 14 days post oocyte retrieval (Week 2) and for women with a positive hCG result, ultrasound was performed at Week 6 post-ET (clinical pregnancy) and at Week 10 post-ET (ongoing pregnancy). Pregnant patients were followed for maternal (adverse events), obstetric (live birth, gestational age at delivery, type of delivery, incidence of twins) and neonatal (sex, weight, height, head circumference, Apgar scores, congenital anomalies, breast feeding, admission to intensive care and specific morbidities e.g. jaundice, respiratory distress syndrome) outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: In an IPD meta-analysis of the clinical trials, a single oral dose of nolasiban 900 mg was associated with an absolute increase of 5.0% (95% CI 0.5, 9.6) in ongoing pregnancy rate and a corresponding increase of 4.4% (95% CI -0.10, 8.93) in live birth rate compared to placebo. Similar magnitude increases were observed for D3 or D5 transfers but were not significantly different from the placebo. Population pharmacokinetics (PK) demonstrated a correlation between higher exposures and pregnancy. LIMITATIONS, REASON FOR CAUTION: The meta-analysis was not a pre-specified analysis. While the individual trials did not show a consistent significant effect, they were not powered based on an absolute increase of 5% in ongoing pregnancy rate. Only a single dose of up to 900 mg nolasiban was administered in the clinical trials; higher doses or extended regimens have not been tested. Only fresh ET has been assessed in the clinical trials to date. WIDER IMPLICATIONS OF THE FINDINGS: The finding support the hypothesis that oxytocin receptor antagonism at the time of ET can increase pregnancy rates following IVF. The overall clinical and population PK data support future evaluation of higher doses and/or alternate regimens of nolasiban in women undergoing ET following IVF. STUDY FUNDING/COMPETING INTERESTS: The trials were designed, conducted and funded by ObsEva SA. A.H., O.P., E.G., E.L. are employees and stockholders of ObsEva SA. E.L. is a board member of ObsEva SA. G.G. reports honoraria and/or non-financial support from ObsEva, Merck, MSD, Ferring, Abbott, Gedeon-Richter, Theramex, Guerbet, Finox, Biosilu, Preglem and ReprodWissen GmbH. C.B. reports grants and honoraria from ObsEva, Ferring, Abbott, Gedeon Richter and MSD. P.P. reports consulting fees from ObsEva. H.T. reports grants and or fees from ObsEva, Research Fund of Flanders, Cook, MSD, Roche, Gedeon Richter, Abbott, Theramex and Ferring. H.V. reports grants from ObsEva and non-financial support from Ferring. P.T. is an employee of Cytel Inc., who provides statistical services to ObsEva. J.D. reports consulting fees and other payments from ObsEva and, Scientific Advisory Board membership of ObsEva. TRIAL REGISTRATION NUMBERS: ClinicalTrials.gov: NCT02310802, NCT03081208, NCT03758885. TRIAL REGISTRATION DATES: December 2014 (NCT02310802), March 2017 (NCT03081208), November 2018 (NCT03758885). FIRST PATIENT'S ENROLMENT: January 2015 (NCT02310802), March 2017 (NCT03081208), November 2018 (NCT03758885).

Expanding the time interval between ovulation triggering and oocyte injection: does it affect the embryological and clinical outcome?

STUDY QUESTION: Is the time interval between ovulation triggering and oocyte denudation/injection associated with embryological and clinical outcome after ICSI? SUMMARY ANSWER: Expanding the time interval between ovulation triggering and oocyte denudation/injection is not associated with any clinically relevant impact on embryological or clinical outcome. WHAT IS KNOWN ALREADY: The optimal time interval between ovulation triggering and insemination/injection appears to be 38-39 h and most authors agree that an interval of >41 h has a negative influence on embryological and clinical pregnancy outcomes. However, in ART centres with a heavy workload, respecting these exact time intervals is frequently challenging. Therefore, we questioned to what extent a wider time interval between ovulation triggering and oocyte injection would affect embryological and clinical outcome in ICSI cycles. STUDY DESIGN, SIZE, DURATION: A single-centre retrospective cohort analysis was performed including 8811 ICSI cycles from 2010 until 2015. Regarding the time interval between ovulation triggering and oocyte injection, seven categories were considered: <36 h, 36 h, 37 h, 38 h, 39 h, 40 h and ≥41 h. In all cases, denudation was performed immediately prior to injection. The main outcome measures were oocyte maturation, fertilization and embryo utilization rate (embryos adequate for transfer or cryopreservation) per fertilized oocyte. Clinical pregnancy rate (CPR) and live birth rate (LBR) were considered as secondary outcomes. Utilization rate, CPR and LBR were subdivided into two groups according to the day of embryo transfer: Day 3 or Day 5. PARTICIPANTS/MATERIALS, SETTING, METHODS: During the study period, oocyte retrieval was routinely performed 36 h post-triggering except in the <36 h group. The interval of <36 h occurred only if OR was carried out before the planned 36 h trigger interval and was followed by immediate injection. Only cycles with fresh autologous gametes were included. The exclusion criteria were: injection with testicular/epididymal sperm, managed natural cycles, conventional IVF, combined conventional IVF/ICSI, preimplantation genetic testing and IVM cycles. Female age, number of oocytes, pre-preparation sperm concentration, post-preparation sperm concentration and motility, day of transfer, number of embryos transferred and quality of the best embryo transferred were identified as potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: Among the seven interval groups, adjusted mean maturation rates ranged from 76.4% to 83.2% and differed significantly (P < 0.001). Similarly, there was a significant difference in adjusted mean fertilization rates (range 69.2-79.3%; P < 0.001). The adjusted maturation and fertilization rates were significantly higher when denudation/injection was performed >41 h post-triggering compared to 38 h post-triggering (reference group). Oocyte denudation/injection at <36 h post-triggering had no significant effect on maturation, fertilization or embryo utilization rates compared to injection at 38 h. No effect of the time interval was observed on CPRs and LBRs, after adjusting for potential confounders. When oocyte injection was performed before 36 h the adjusted analysis showed that compared to 38 h after ovulation triggering the chance of having a live birth tends to be lower although the difference was not statistically significant (odds ratio 0.533, 95% CI: 0.252-1.126; P = 0.099). Injection ≥41 h post-triggering did not affect LBR compared to injection at 38 h post-ovulation. LIMITATIONS, REASONS FOR CAUTION: As this is a large retrospective study, the influence of uncontrolled variables cannot be excluded. These results should not be extrapolated to other ART procedures such as IVM, conventional IVF or injection with testicular/epididymal sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that the optimal injection time window may be less stringent than previously thought as both embryological and clinical outcome parameters were not significantly affected in our analysis. This is reassuring for busy ART centres that might not always be able to follow strict time intervals. STUDY FUNDING/COMPETING INTEREST(S): No funding. The authors declare no conflict of interest related to the present study. TRIAL REGISTRATION NUMBER: N/A.

Early pregnancy loss in patients with polycystic ovary syndrome after IVM versus standard ovarian stimulation for IVF/ICSI.

STUDY QUESTION: Is the incidence of early pregnancy loss (EPL) in patients with polycystic ovary syndrome (PCOS) higher after IVM of oocytes than after ovarian stimulation (OS) for IVF/ICSI? SUMMARY ANSWER: Women with PCOS who are pregnant after fresh embryo transfer have a higher probability of EPL following IVM, but after frozen embryo transfer (FET), no significant difference in the incidence of EPL was observed following IVM compared to OS. WHAT IS KNOWN ALREADY: There is conflicting evidence in the current literature with regard to the risk of EPL after IVM of oocytes when compared with OS. Because of the limited sample size in previous studies, the use of different IVM systems and the possible bias introduced by patient characteristics and treatment type, firm conclusions cannot be drawn. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study evaluating 800 women, with a diagnosis of infertility and PCOS as defined by Rotterdam criteria, who had a first positive pregnancy test after fresh or FET following IVM or OS between January 2010 and December 2017 in a tertiary care academic medical centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: Pregnancies after non-hCG triggered IVM following a short course of highly purified human menopausal gonadotropin were compared with those after conventional OS. The primary outcome was EPL, defined as a spontaneous pregnancy loss before 10 weeks of gestation. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 329 patients with a positive pregnancy test after IVM and 471 patients with a positive pregnancy test after OS were included. Women who were pregnant after IVM were younger (28.6 ± 3.4 years vs 29.3 ± 3.6 years, P = 0.005) and had higher serum anti-Mullerian hormone levels (11.5 ± 8.1 ng/ml vs 7.2 ± 4.1 ng/ml, P < 0.001) compared to those who were pregnant after OS. The distribution of PCOS phenotypes was significantly different among women in the IVM group compared to those in the OS group and women who were pregnant after OS had previously suffered EPL more often (28% vs 17.6%, P = 0.003). EPL was significantly higher after fresh embryo transfer following IVM compared to OS (57/122 (46.7%) vs 53/305 (17.4%), P < 0.001), while the results were comparable after FET (63/207 (30.4%) vs 60/166 (36.1%), respectively, P = 0.24). In the multivariate logistic regression analysis evaluating fresh embryo transfer cycles, IVM was the only independent factor (adjusted odds ratio (aOR) 4.24, 95% CI 2.44-7.37, P < 0.001)) significantly associated with increased odds of EPL. On the other hand, when the same model was applied to FET cycles, the type of treatment (IVM vs OS) was not significantly associated with EPL (aOR 0.73, 95% CI 0.43-1.25, P = 0.25). LIMITATIONS, REASONS FOR CAUTION: The current data are limited by the retrospective nature of the study and the potential of bias due to unmeasured confounders. WIDER IMPLICATIONS OF THE FINDINGS: The increased risk of EPL after fresh embryo transfer following IVM may point towards inadequate endometrial development in IVM cycles. Adopting a freeze-all strategy after IVM seems more appropriate. Future studies are needed to ascertain the underlying cause of this observation. STUDY FUNDING/COMPETING INTEREST(S): The Clinical IVM research has been supported by research grants from Cook Medical and Besins Healthcare. All authors declared no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Combining fertility preservation procedures to spread the eggs across different baskets: a feasibility study.

STUDY QUESTION: What is the reproductive potential following combinations of ovarian stimulation, IVM and ovarian tissue cryopreservation (OTC) in female patients seeking fertility preservation (FP)? SUMMARY ANSWER: In selected patients, combining different FP procedures is a feasible approach and reproductive outcomes after FP in patients who return to attempt pregnancy are promising. WHAT IS KNOWN ALREADY: FP is increasingly performed in fertility clinics but an algorithm to select the most suitable FP procedure according to patient characteristics and available timeframe is currently lacking. Vitrification of mature oocytes (OV) and OTC are most commonly performed, although in some clinical scenarios a combination of procedures including IVM, to spread the sources of gametes, may be considered in order to enhance reproductive options for the future. STUDY DESIGN, SIZE, DURATION: Retrospective, observational study in a university-based, tertiary fertility centre involving all female patients who underwent urgent medical FP between January 2012 and December 2018. Descriptive analysis of various FP procedures, either stand-alone or combined, was performed, and reproductive outcomes of patients who attempted pregnancy in the follow-up period were recorded. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 207 patients underwent medical FP. Patient-tailored strategies and procedures were selected after multidisciplinary discussion. When deemed feasible, FP procedures were combined to cryopreserve different types of reproductive tissue for future use. The main primary outcome measure was the number of mature oocytes. Live birth rates were evaluated in patients who returned for reproductive treatment. MAIN RESULTS AND THE ROLE OF CHANCE: Among patients seeking FP, 95/207 (46%) had breast cancer, 43/207 (21%) had haematological malignancies and 31/207 (15%) had a gynaecological tumour. Mean ± SD age was 27.0 ± 8.3 years. Eighty-five (41.1%) patients underwent controlled ovarian stimulation (COS), resulting in 10.8 ± 7.1 metaphase II (MII) oocytes for vitrification. Eleven (5.3%) patients had multiple COS cycles. Transvaginal oocyte retrieval for IVM was performed in 17 (8.2%) patients, yielding 9.2 ± 10.1 MII oocytes. Thirty-four (16.4%) patients underwent OTC combined with IVM of oocytes retrieved from ovarian tissue 'ex vivo' (OTO-IVM), yielding 4.0 ± 4.3 MII oocytes in addition to ovarian fragments. Seventeen (8.2%) patients had OTC combined with OTO-IVM and transvaginal retrieval of oocytes for IVM from the contralateral ovary, resulting in 13.5 ± 9.7 MII oocytes. In 13 (6.3%) patients, OTC with OTO-IVM was followed by controlled stimulation of the contralateral ovary, yielding 11.3 ± 6.6 MII oocytes in total. During the timeframe of the study, 31/207 (15%) patients have returned to the fertility clinic with a desire for pregnancy. Of those, 12 (38.7%) patients had preserved ovarian function and underwent ART treatment with fresh oocytes, resulting in nine (75%) livebirth. The remaining 19 (61.3%) patients requested warming of their cryopreserved material because of ovarian insufficiency. Of those, eight (42.1%) patients had a livebirth, of whom three after OTO-IVM. To date, 5/207 patients (2.4%) achieved an ongoing pregnancy or livebirth after spontaneous conception. LIMITATIONS, REASONS FOR CAUTION: Our FP programme is based on a patient-tailored approach rather than based on an efficiency-driven algorithm. The data presented are descriptive, which precludes firm conclusions. WIDER IMPLICATIONS OF THE FINDINGS: Combining different FP procedures is likely to enhance the reproductive fitness of patients undergoing gonadotoxic treatment but further follow-up studies are needed to confirm this. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used for this study and the authors have no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Chromosomal abnormalities after ICSI in relation to semen parameters: results in 1114 fetuses and 1391 neonates from a single center.

STUDY QUESTION: Is there a relationship between karyotype abnormalities in fetuses and children conceived by ICSI and their father's semen parameters? SUMMARY ANSWER: The de novo chromosomal abnormality rate in pre- and postnatal karyotypes of ICSI offspring was higher than in the general population and related to fathers' sperm parameters. WHAT IS KNOWN ALREADY: Several studies have reported a higher rate of de novo chromosomal anomalies in ICSI fetuses but recent data from large cohorts are limited. Overall, reported prevalences of non-inherited karyotype aberrations are increased in fetuses conceived after ICSI and vary between 1.6% and 4.2%. Only a few studies focus on the relation between karyotype anomalies in ICSI offspring and semen parameters of their fathers. Furthermore, an increased incidence of abnormal karyotypes in ICSI neonates has been described, but the rates vary widely across studies. STUDY DESIGN, SIZE, DURATION: We report on karyotype results from prenatal testing by means of chorionic villus sampling and amniocentesis and results from postnatal blood sampling in offspring conceived by ICSI in a single center. Ongoing pregnancies resulting from an oocyte retrieval between January 2004 and December 2012 and after transfer of fresh ICSI embryos obtained using ejaculated or non-ejaculated sperm (fresh or frozen-thawed) were considered. Pregnancies following frozen embryo transfer, oocyte or sperm donation, IVF, preimplantation genetic testing and IVM were excluded. All abnormal prenatal results after sampling are reported irrespective of the outcome of the pregnancy. PARTICIPANTS/MATERIALS, SETTING, METHODS: From the 4816 ongoing ICSI pregnancies, information on pregnancy outcome was available for 4267 pregnancies. Prenatal testing was performed in 22.3% of the pregnancies, resulting in a diagnosis in 1114 fetuses. A postnatal karyotype was obtained in 29.4% of the pregnancies in which no invasive prenatal diagnosis was performed, resulting in a total of 1391 neonates sampled. The prevalence of chromosomal anomalies according to maternal age and semen quality was analyzed with logistic regression. For definitions of normal semen quality, the World Health Organization reference values for human semen characteristics were adopted. MAIN RESULTS AND THE ROLE OF CHANCE: An abnormal fetal karyotype was found in 29 singletons and 12 multiples (41/1114; 3.7%; 95% CI 2.7-4.9%): 36 anomalies were de novo (3.2%; 95% CI 2.3-4.4), either numerical (n = 25), sex (n = 6) or structural (n = 5), and five were inherited. Logistic regression analysis did not show a significant association between maternal age and a de novo chromosomal fetal abnormality (odds ratio (OR) 1.05; 95% CI 0.96-1.15; P = 0.24). In all but one case, fetuses with an abnormal karyotype were conceived by ICSI using ejaculated sperm.Abnormal karyotypes were found in 14 (1.0%; 95% CI 0.6-1.7) out of 1391 postnatal samples of children born after ICSI who were not tested prenatally: 12 were de novo anomalies and two were inherited balanced karyotypes. The 14 abnormal karyotypes were all found in children born after ICSI using ejaculated sperm.The odds of a de novo karyotype aberration increased with maternal age when combining pre- and postnatal data (OR 1.11; 95% CI 1.04-1.19). A higher rate of de novo chromosomal abnormalities was found in fetuses and children of couples with men having a sperm concentration <15 million/ml (adjusted OR (AOR) 2.10; 95% CI 1.14-3.78), sperm concentration <5 million/ml (AOR 1.9; 95% CI 1.05-3.45) and total sperm count <10 million (AOR 1.97; 95% CI 1.04-3.74). LIMITATIONS, REASONS FOR CAUTION: We cannot exclude that the observation of a higher prevalence of karyotype anomalies in ICSI offspring compared to literature data in the general population is due to enhanced surveillance after ART given the lack of a control group. Although we did not find more chromosomal anomalies after ICSI with non-ejaculated sperm, the small numbers do not allow firm conclusions. WIDER IMPLICATIONS OF THE FINDINGS: The observed increased risk of a de novo karyotype anomaly after ICSI conception in couples with poor sperm warrants continued counseling toward prenatal testing.The current and widespread use of innovative non-invasive prenatal testing will result in larger datasets, adding to a balanced estimation of the prevalence of karyotype anomalies in ICSI offspring. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Methusalem grants issued by the Vrije Universiteit Brussel. All authors declared no conflict of interest related to this study. TRIAL REGISTRATION NUMBER: N/A.