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.

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.

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.