Follicular flushing leads to higher oocyte yield in monofollicular IVF: a randomized controlled trial.

STUDY QUESTION: Does follicular flushing increase the number of mature oocytes in monofollicular IVF? SUMMARY ANSWER: Follicular flushing increases the number of mature oocytes in monofollicular IVF. WHAT IS KNOWN ALREADY: Flushing increases neither the oocyte yield nor the pregnancy rate in polyfollicular IVF or in poor responder patients. In monofollicular IVF, the effect of flushing has so far been addressed by two studies: (i) a prospective study with minimal stimulation IVF demonstrated an increased oocyte yield, and (ii) a retrospective study with natural cycle (NC)-IVF showed an increased oocyte yield and an increased transfer rate. STUDY DESIGN, SIZE, DURATION: Randomized controlled trial including 164 women who were randomized for either aspiration with or without flushing from 2016 to 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile women 18-42 years of age with an indication for IVF treatment at a university-based infertility unit. Women undergoing monofollicular IVF were randomized to either follicular aspiration only or follicular aspiration directly followed by five follicular flushes at a 1:1 ratio. The intervention was done without anaesthesia, using a gauge 19 single-lumen needle. Flushing volume was calculated (sphere formula) based on the size of the follicle. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 164 women were included; 81 were allocated to 'aspiration only' and 83 to additional 'flushing'. Primary analysis was based on the intention-to-treat: oocyte yield, defined as the collected mature oocyte rate, was higher (n = 64/83, 77.1%) in the flushing group compared to the aspiration only group (n = 48/81, 59.3%, adjusted risk difference (RD): 18.2% (95% CI 3.9-31.7%), P-value = 0.02). In the flushing group, most oocytes were retrieved within the first three flushes (63/83, 75.8%). Fertilization rate was higher in the flushing group (n = 53/83, 63.9% vs n = 38/81, 46.9%; adjusted RD: 16.8% (96% CI 1.5-31.4%), P = 0.045). Transfer rate was also higher in the flushing group (n = 52/83, 62.7% vs n = 38/81, 46.9%; RD: 15.71 (95% CI 0.3-30.3%)), but the difference was not significant (P = 0.06). The clinical pregnancy rate n = 9/83 versus n = 9/81 (RD: -0.3% (95% CI -9.9% to 9.5%)) and live birth rate n = 7/83 versus n = 8/81 (RD: -1.5% (95% CI -10.4% to 7.1%)) were not significantly different between the flushing and the aspiration group. The median duration of the intervention was significantly longer with flushing (2.38 min; quartiles 2.0, 2.7) versus aspiration only (0.43 min; quartiles 0.3, 0.5) (P < 0.01). There was no significant difference in the mean (±SD) visual analogue scales pain score between the follicular flushing (3.4 ± 1.8) and the aspiration group (3.1 ± 1.89). LIMITATIONS, REASONS FOR CAUTION: Blinding of the procedure was not possible. WIDER IMPLICATIONS OF THE FINDINGS: Our study proved that flushing of single follicles in NC-IVF increases the oocyte yield. In contrast to polyfollicular IVF flushing seems to be beneficial in a monofollicular setting if the technique used in our study (single-lumen needle, 5 flushings with flushing volume adaptation) is applied. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the financial sources of the division and in part by a research grant provided by NMS Biomedical SA, Switzerland. The company did not have any roles in design or conduct of the study or in the preparation of the manuscript. The authors have no other conflicts of interest. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov NCT02641808. TRIAL REGISTRATION DATE: 29 December 2015. DATE OF FIRST PATIENT'S ENROLMENT: 22 August 2016.

Individual spatial aggregation correlates with between-population variation in fine-scale genetic structure of Silene ciliata (Caryophyllaceae).

Fine-scale genetic structure (FSGS) can vary among populations within species depending on multiple demographic and environmental factors. Theoretical models predict that FSGS should decrease in high-density populations and increase in populations where individuals are spatially aggregated. However, few empirical studies have compared FSGS between populations with different degrees of individual spatial aggregation and microhabitat heterogeneity. In this work, we studied the relationship between spatial and genetic structure in five populations of alpine specialist Silene ciliata Poiret (Caryophyllaceae). We mapped all individuals in each population and genotyped 96 of them using 10 microsatellite markers. We found significant FSGS consistent with an isolation-by-distance process in three of the five populations. The intensity of FSGS was positively associated with individual spatial aggregation. However, no association was found between FSGS and global population density or microhabitat heterogeneity. Overall, our results support theoretical studies indicating that stronger spatial aggregation tends to increase the magnitude of FSGS. They also highlight the relevance of characterizing local plant distribution and microhabitat to better understand the mechanisms that generate intraspecific variation in FSGS across landscapes.

How to escape from crop-to-weed gene flow: phenological variation and isolation-by-time within weedy sunflower populations.

The evolution of crop-related weeds may be constrained by recurrent gene flow from the crop. However, flowering time variation within weedy populations may open the way for weed adaptation by allowing some weeds to escape from this constraint. We investigated this link between phenology, gene flow and adaptation in weedy sunflower populations that have recently emerged in Europe from crop-wild hybridization. We studied jointly flowering phenology and genetic diversity for 15 microsatellite loci in six cultivated sunflower (Helianthus annuus) fields infested by weedy sunflower populations. The flowering overlap of cultivated and weedy sunflowers varied between and within populations: some weedy individuals were found to be completely isolated from the crop, the frequency of these plants being higher in populations from highly infested fields. Within weedy populations, we detected a pattern of isolation-by-time: the genetic divergence between individuals was positively correlated with their divergence in flowering period. In addition, earlier weeds, which flowered synchronously with the crop, were genetically more similar than late-flowering weeds to the cultivated varieties. Overall, our results suggest that crop-to-weed gene flow occurred, but was limited by divergent phenologies. We discuss the roles of weed adaptation and population history in the generation of this partial reproductive isolation.

Estimation of mating system parameters in an evolving gynodioecous population of cultivated sunflower (Helianthus annuus L.).

Cultivated plants have been molded by human-induced selection, including manipulations of the mating system in the twentieth century. How these manipulations have affected realized parameters of the mating system in freely evolving cultivated populations is of interest for optimizing the management of breeding populations, predicting the fate of escaped populations and providing material for experimental evolution studies. To produce modern varieties of sunflower (Helianthus annuus L.), self-incompatibility has been broken, recurrent generations of selfing have been performed and male sterility has been introduced. Populations deriving from hybrid-F1 varieties are gynodioecious because of the segregation of a nuclear restorer of male fertility. Using both phenotypic and genotypic data at 11 microsatellite loci, we analyzed the consanguinity status of plants of the first three generations of such a population and estimated parameters related to the mating system. We showed that the resource reallocation to seed in male-sterile individuals was not significant, that inbreeding depression on seed production averaged 15-20% and that cultivated sunflower had acquired a mixed-mating system, with ∼50% of selfing among the hermaphrodites. According to theoretical models, the female advantage and the inbreeding depression at the seed production stage were too low to allow the persistence of male sterility. We discuss our methods of parameter estimation and the potential of such study system in evolutionary biology.

Clomiphene citrate stimulated cycles - additional gonadotrophin stimulation increases endometrium thickness without increasing implantation rate.

It is known that Clomiphene citrate (CC) reduces endometrial thickness, but it is unknown if additional gonadotrophin stimulation increases endometrial thickness and if this has an effect on implantation rate in in vitro fertilization (IVF). The retrospective study included 263 minimal stimulation IVF-cycles stimulated with 25 mg CC per day (CC-IVF), and 161 IVF-cycles stimulated with CC plus 75IU hMG (human Menopausal Gonadotrophin) per day (CC/hMG-IVF). Endometrial and oestradiol (E2) measurements were analysed between day -4 and 0 (0 = day of oocyte retrieval) and the association of endometrial thickness and treatment on implantation rates were studied after multiple adjustments. It was shown that on day 0, endometrium was significantly thicker in CC/hMG-IVF versus CC-IVF cycles (9.81 ±2.68 versus 9.06 ±2.54 mm, p = 0.005). However, increased endometrial thickness did not have an effect on implantation and live birth rates. In conclusion, gonadotrophins should not be added to low dose CC treated IVF cycles just to increase endometrial thickness as increased endometrial thickness does not increase implantation rate.