Improve IVF Outcomes Research Articles

Subcutaneous G-CSF administration improves IVF outcomes in patients with recurrent implantation failure presenting a KIR/HLA-C mismatch

Research questionDespite advances in assisted reproductive technologies, many blastocysts are lost unexpectedly during implantation. Alterations in maternal immune tolerance towards fetal antigens may contribute to adverse IVF outcomes. The purpose of this study is to evaluate whether administering Granulocyte Colony-Stimulating Factor (G-CSF) to couples with a Human Leukocyte Antigen/Killer-Cell Immunoglobulin-Like Receptor (HLA/KIR) mismatch could positively modulate the implantation process in patients with recurrent implantation failure (RIF). A KIR/HLA-C mismatch occurs when the interaction between KIRs and HLA-C causes an inhibition of NK cells, which may result in reduced G-CSF secretion leading to impaired placentation and increased risk of miscarriage, pre-eclampsia and fetal growth restriction. DesignA retrospective monocentric cohort study conducted at the IVI Clinic in Rome, including women with a history of at least two failed blastocyst transfers. Couples underwent KIR and HLA-C testing. Couples with a KIR/HLA-C mismatch received G-CSF subcutaneously up to week nine of gestation. The mismatch included cases with inhibitory KIR genotypes and HLA-C2C2 females with HLA-C1C1, or C1C2 males or HLA-C1C2 females with male HLA-C2C2. The reproductive outcomes were assessed, and the logistic regression models controlled for potential confounders affecting IVF outcomes. Results79 patients with RIF and a KIR/HLA-C mismatch were included in the study. 30 patients were administered G-CSF, and 49 received no treatment. In the univariate analysis, no statistically significant differences were reported in the reproductive outcomes after IVF between the women treated with G-CSF and the control group. However, the logistic regression analysis that controlled for confounding factors showed that patients treated with subcutaneous G-CSF had statistically significant higher ongoing-pregnancy (aOR=3.808) and live-birth (aOR=4.998) rates, and a lower miscarriage rate (aOR=0.057). No statistically significant differences were found in other reproductive outcomes. ConclusionThe use of subcutaneous G-CSF in patients with a KIR/HLA-C mismatch undergoing IVF may reduce miscarriage and improve live-birth rates. G-CSF may modulate NK-mediated immune mechanisms and improve trophoblast invasion and development. Randomized trials are warranted to validate these findings and enhance the chances of successful pregnancies in couples with an immunological mismatch.

O-056 Can we tailor ART to endometriosis patients?

Abstract ART remains an effective treatment for endometriosis-associated infertility, although there is evidence that pregnancy rates are diminished in women with endometriosis compared with other etiologies of infertility. In this lecture, the literature relating to endometriosis-associated infertility will be evaluated and recommendations will be made on the management of patients both pre-ART and during ART in order to improve ART outcomes in women with endometriosis. Surgery as an adjuvant to ART appears to have a favorable effect in patients with minimal to mild endometriosis prior to IVF. However, this evidence is based on only one study and more research is needed. As deep endometriosis (DE) is usually accompanied by advanced intra-abdominal disease resulting in distortion of the pelvic anatomy and tubal dysfunction, it is not surprising that IVF is considered as first line treatment. Observational data are available on the outcomes of DE surgery regarding conception rates in infertile patients with endometriosis suggesting that surgery may increase natural pregnancy rates as well as improve IVF outcomes. However, randomized trials comparing IVF to DE surgery are non-existent, as DE surgery is still mainly performed for pain and reduced quality of life rather than for treating infertility. In order to improve IVF success rates in endometriosis, various pre-ART treatments have been suggested. The oldest one relates to the use of gonadotropin releasing hormone (GnRH) agonist prior to IVF. Currently there is uncertainty as to whether long-term GnRH agonist therapy is beneficial when compared to standard IVF/ICSI in endometriosis. In addition, there is no evidence to support the use of oral GnRH antagonists and oral contraceptives as pre-treatment prior to ART, but results of ongoing studies will determine further. More recent studies support defects in endometrial receptivity as a cause of IVF failure. In recent years a new marker for endometrial receptivity in endometriosis emerged: BCL6, a biomarker for endometrial inflammation as it stimulates endometrial cytokine expression. Prospective cohort data provide a proof of concept that high BCL6 expression is associated with adverse IVF outcomes in women with endometriosis and that patients with high BCL6 expression may benefit from medical and surgical treatment prior to IVF. In order to improve IVF success rates in endometriosis, various pre-ART treatments have been suggested. The oldest one relates to the use of gonadotropin releasing hormone (GnRH) agonist prior to IVF. Contrary to previous findings, there is currently uncertainty as to whether long-term GnRH agonist therapy is beneficial when compared to standard IVF in endometriosis. In addition, there is also no evidence to support the use of oral GnRH antagonists and oral contraceptives as pre-treatment prior to ART in endometriosis, but results of ongoing studies will determine further. It has been hypothesized that applying local endometrial injury might induce a beneficial effect on endometrial receptivity prior to ART. However, scratching the endometrium as well as infusing fluids (Lipiodol and ExEm gel) into the uterine cavity (uterine bathing) in endometriosis patients prior to ART did not improve IVF success. During ART, a specific protocol for ART in women with endometriosis cannot be recommended. Both GnRH agonist and antagonist protocols can be used. For ovarian stimulation, a higher dose of gonadotropins may be required in stage III-IV endometriosis due to a poorer ovarian response. There is insufficient evidence for the routine use of prophylactic antibiotics at the time of oocyte retrieval in patients with endometriosis, but their use is recommended in patients with endometrioma. Considering fertilization, there appears no added benefit in using ICSI over IVF in endometriosis unless male-factor infertility is present. Finally, there is insufficient evidence to recommend any particular luteal support in women with endometriosis undergoing ART.

P-013 A potential therapeutic approach of intermittent fasting on sperm viscosity

Abstract Study question Does intermittent fasting affect sperm viscosity and motility? Summary answer The obtained results demonstrate that intermittent fasting significantly improves sperm viscosity and motility, and might be considered as a potential therapeutic approach for sperm hyperviscosity. What is known already Numerous factors influence sperm quality, functionality, and, consequently, male fertility. In the last decade, nutritional deficiency and lifestyle, have been grouped as popular factors that affect human sperm function and fertilization processes. Semen hyperviscosity which occurs in 12-29% of ejaculates is another condition that can contribute to male subfertility, as it is associated with changes in the chemical and physical characteristics of semen. Additionally, semen hyperviscosity contributes as well to poor sperm motility. Based on the aforementioned facts, focusing on identifying factors to enhance sperm viscosity and motility is crucial for improving IVF outcomes. Study design, size, duration The present study was based on semen analyses of 60 samples from 30 IVF patients. Following the purpose of the study and inclusion criteria, all 30 patients were diagnosed with abnormal sperm viscosity. Out of this number, 13 patients had asthenozoospermia, 4 patients were diagnosed with asthenoteratozoospermia, 7 patients were diagnosed with teratozoospermia and 6 patients had normozoospermia. All patients followed 4 days of sexual abstinence. All specimens were analyzed within 1 hour of collection. Participants/materials, setting, methods Sperm analysis including sperm viscosity was processed according to the WHO laboratory manual sperm criteria (Sixth edition 2021). For the study purpose, all the participants followed an intermittent fasting period of 14-16 hours for 30 days. Samples were analyzed twice, the first time before fasting and the second time after 30 days of fasting. Most of the patients did not consume food from 6 pm until 8 or 10 am. Main results and the role of chance The average age of the patients included in the study was 37.53 ± 4.6 years, while the BMI ranged from 21.26 to 32.28 kg/m2. Abnormal viscosity (hyperviscosity) was diagnosed when the drop formed a thread more than 2 cm long, while normal liquefied ejaculate fell as small discrete drops. The obtained results indicated that sperm parameters were improved after intermittent fasting. The most significant difference was observed in semen viscosity where out of the 30 patients who were diagnosed with abnormal semen viscosity after 30 days of intermittent fasting only 9 patients still had viscose semen (p<0.0001). Additionally, a significant difference was revealed in sperm motility before and after the fasting period as well(33.73±12.15 vs 44.10 ±11.54; respectively p < 0.001). Among the 17 subjects initially diagnosed with abnormal motility, following the implementation of intermittent fasting, 11 demonstrated a sperm improvement and were diagnosed with normal motility. The difference in sperm viability (52.8± 7.71 vs 61.3±8.37; respectively p < 0.01) and Ph values before and after fasting as well were statistically significant. On the other side, although sperm concentration (Mil/ml) was increased after fasting, the difference was not statistically significant (39.03±19.52 vs 41.53±19.19; respectively p = 0.92). Additionally, intermittent fasting did not affect sperm morphology either. Limitations, reasons for caution The limitation of the presented study was a relatively small patient cohort. Further research should link intermittent fasting with IVF outcomes. Wider implications of the findings Until today there are no clear dietary guidelines created for infertile males. As far as we know this is the first research, that includes a comparison of sperm parameters, based on intermittent fasting, and the obtained results confirm that intermittent fasting can be used as a potential therapeutic approach. Trial registration number not applicable

O-031 Tacrolimus treatment significantly improved IVF outcomes with euploid embryo transfer in women with repeated implantation failures (RIF) and elevated T helper (Th)1/Th2 cell ratios

Abstract Study question Dose tacrolimus improve implantation rate for RIF women showing elevated T helper (Th)1/Th2 cell ratios, particularly those undergoing IVF/ET with euploid blastocysts? Summary answer Tacrolimus improved the implantation rate in RIF women with elevated Th1/Th2 cell ratios undergoing IVF/ET using euploid blastocysts. What is known already The peripheral blood Th1 and Th2 cells determine immune inflammatory responses at the maternal-fetal interface. Previously, we reported that about 40% of women with RIF (≥4) had elevated Th1/Th2 cell ratios (≥10.3). The pregnancy rate was significantly improved when they were treated with tacrolimus, an immunosuppressive agent [AJRI 2015]. However, genetic embryonic concerns of RIF could not be ruled out since not all transferred embryos were genetically screened. Recently, we reported an adjusted cut-off value of the Th1/Th2 cell ratios (≥11.8) [AJRI 2021] for women with RIF based on the retrospective analysis. However, the prospective study has not been performed. Study design, size, duration A prospective cohort study was performed, including 569 infertile women with more than 4 times of RIF. The study participants received ET with euploid blastocysts from September 2020 to November 2021. All transferred blastocysts were confirmed as euploid or low-frequency mosaic by preimplantation genetic testing for aneuploidy, and the endometrial preparation for ET was made by using either hormone replacement cycle or natural ovulatory cycle. Participants/materials, setting, methods Before ET, all participants were measured for their peripheral blood Th1/Th2 (CD4+IFN-γ+/ CD4+IL-4+) cell ratios in the secretory phase. Women who had elevated Th1/Th2 cell ratios (≥10.3) received tacrolimus (2-4 mg daily). Tacrolimus was started 2 days before ET, and the hCG and gestational sac (GS) rates were calculated in women with or without tacrolimus. Main results and the role of chance Of 569 participants, 174 (30.6%) women showed elevated Th/Th2 cell ratios (≥10.3), whereas the remaining 395 women served as control (Control group). Among 174 women, 148 women received tacrolimus (Tac group), while 26 did not receive tacrolimus (no-Tac group). The hCG and GS rates of the Tac group were 73.0% and 64.2%, respectively, similar to the no-Tac group (69.2% and 57.7%, respectively, P=NS). Meanwhile the hCG and GS rates of the control group were 70.6% and 60.0%, respectively (P=NS). When the Th1/Th2 cell ratio cut-off value was set to ≥ 11.8, 123 women of the 569 participants (21.6%) had elevated Th1/Th2 cell ratios (≥11.8). Among them, 112 received tacrolimus (Tac2 group), and 11 did not tacrolimus (no-Tac2 group). The remaining 410 women were as a control group (Control2 group). The GS rate of the Tac2 group was 67.0%, which was significantly higher than that of the no-Tac group (36.3%, p < 0.05), while the GS rate of the Control2 group was 60.5% (P=NS, vs Control2 group). Tacrolimus dosing data was as follows; 11.8 or more but less than 15.8 of Th1/Th2 cell ratios were needed for 2 mg of tacrolimus daily, and 15.8 pr more was needed for 3 mg of tacrolimus. Limitations, reasons for caution The study was a controlled trial with a limited number of study population. Additionally, the endometrial changes or peripheral immune responses after tacrolimus were not evaluated thoroughly. Further study is needed for the tacrolimus effect on systematic immune responses and local endometrial immune milieu. Wider implications of the findings Before ET, screening for increased Th1/Th2 ratios (Cut-off value ≥11.8) may significantly improve IVF outcomes by selecting proper candidates for tacrolimus treatment and detecting the proper tacrolimus doses. Notably, a significant proportion of women with RIF have additional implantation failures, even with euploid embryos, when immune-inflammatory conditions were not controlled. Trial registration number not applicable

P-764 A decade of transformations in IVF strategies towards enhanced clinical efficacy, efficiency, and safety: the real-life experience of 6610 couples

Abstract Study question To what extent do clinical and laboratory strategies affect the main measures of IVF success? Summary answer Across 10 years, the implementation and increasing adoption of antagonist protocols combined with agonist-trigger, freeze-all, blastocyst culture, PGT, and multicycle counseling approach improved IVF outcomes. What is known already The primary indicator of IVF success is the cumulative-live-birth-rate per intention-to-treat (CLBR per ITT). Since reproductive competence is inherent to every gamete and embryo, IVF treatments cannot guarantee success. However, they can maximize the likelihood of success with the safest approaches available. Furthermore, there are other outcomes to consider for a more thorough assessment of IVF effectiveness and efficiency, including time in treatment (=LB achieved / no LB achieved and no embryo available for transfer; TiT), miscarriage rate, and prevalence of multiple-births. Moreover, a family planning perspective should be strengthened in IVF aiming at ≥ 2 healthy children, whenever feasible. Study design, size, duration Longitudinal observation of 6610 couples undergoing ≤6 ovarian-stimulations (OS) for IVF at a private clinic within 3 years. Couples were clustered in 11 groups according to the year of their first treatment (years: 2010-2020) and compared for (i) CLBR within 3 years, (ii) prevalence of ≥ 1 miscarriage and ≥1 twin-delivery, (iii) TiT (from first-OS to the last IVF procedure before treatment conclusion), (iv) prevalence of ≥ 2 singleton-deliveries within 6 years. Participants/materials, setting, methods The naïve patients included increased from ≈500 in 2010 to ≥ 600 between 2013 and 2020. The maternal age was stable around 38±4 years with ≈20% prevalence of severe male factor. OS was conducted through long, short flare-up, mild, antagonist, progestin-primed, or DuoStim protocols with hCG/agonist-trigger. All patients underwent ICSI on fresh own eggs and cleavage/blastocyst culture, fresh transfer/freeze-all, untested/euploid transfers, single/multiple-embryo transfers. Main results and the role of chance Antagonist-protocols (≈20% in 2010 and ≈80% in 2020) combined with agonist-trigger (0.4% and 76.8%) superseded long/short flare-up (≈60% and <10%) and hCG-trigger (99.6% and 23.2%). DuoStim, introduced in 2015, reached 15%-prevalence by 2020. The MII-oocytes collected at first-retrievals increased from 5±3 (2010) to 7.2±4.9 (2020). The prevalence of first-cycles without embryos raised from 3% to 14% by 2020 primarily due to an evident cleavage-to-blastocyst culture switch (2010:96% versus 4%; 2020:1% versus 99%). Similarly, PGT-A and freeze-all utilization respectively increased from 3% to 80% and from 12% to 93%. Although TiT increased from ≈2 cycles in ≈140 days (2010-2013) to ≈1.6 in ≈160 days (2014-2020), the 3-years CLBR improved from 32% (2010) to 42% (2020), a difference confirmed also adjusting for maternal age and overall number of MII-oocytes collected (multivariate-OR:1.03,95%CI:1.01-1.05;adjusted-p=0.001). This was paralleled by the halving of women suffering ≥1 miscarriage (≈12% to < 6%;p=0.002) and drop in women with ≥1 twin-delivery (7.5% to 0.5%;p<0.001). The latter achievement was due to single-embryo transfer rates increasing from 26% (2010) to 98% (2020). Accordingly, the women starting in 2017 and obtaining ≥2 LBs within 6-years were 9.9%, 98% of which after consecutive singleton-deliveries; these figures were 6.6% and just 22% among women starting in 2010(p = 0.004). Limitations, reasons for caution This study suffers from the inherent limitations of a retrospective design. Temporal changes, other than clinical strategies and laboratory advancements (e.g., personnel), might have influenced the results, although no critical infrastructural changes were implemented. Gestational and neonatal follow-up data are missing in this study. Wider implications of the findings Clinical and laboratory advancements enhanced IVF efficacy and efficiency over time also fulfilling family planning desire. Future strategies and improved workflows should focus on reducing drop-out and time-to-LB. As larger oocyte cohorts led to higher CLBR throughout maternal age, tailored-multicycle approach might represent a valuable option to this end. Trial registration number none

O-059 ART in PCOS

Abstract Polycystic Ovarian Syndrome affects an estimated 8 – 13% of women of reproductive age (WHO). It is the commonest cause of anovulation and infertility. The high AMH, LH, Insulin resistance and hyperandrogenism seen in PCOS women contributes to infertility along with poor oocyte quality and reduced endometrial receptivity. ART procedures are recommended as the third line of treatment after ovulation induction, IUI or ovarian drilling. Prior to IVF, metabolic assessment and lifestyle adjustments with weight management must be done. Adjuvant agents such as metformin melatonin, myoinositol and Vitamin-D have been tried to improve IVF outcomes. Mild stimulation is an option in patients as high risk for ovarian hyperstimulation syndrome (OHSS). Conventional stimulation with low dose FSH, GnRH antagonist down regulation, agonist trigger with freezing of all embryos, significantly reduces risk of OHSS without compromising cumulative live birth rates. Adjustments for stimulation protocols may need to be made for BMI, insulin resistance and AMH. In vitro maturation can be considered in patients with prior severe OHSS, though the results may be inferior. ART in PCOS patients is challenging and even after pregnancy is achieved, there may be a higher risk of adverse obstetric outcomes.

P-190 Impact of low lactate culture media in embryo development and IVF outcomes

Abstract Study question Can low lactate culture media improve embryo utilization rate in artificial reproductive technology (ART) cycles? Summary answer Embryos cultured in low lactate media showed a significant improved blastocyst utilization rate compared to embryos cultured in standard culture media. What is known already Culture media plays a key role in the embryonic development in the IVF lab. Currently, different culture medias are commercially available, yet no consensus on the most suitable media composition has been reached. Recently, minimizing metabolic stress for the embryo to enhance implantation and pregnancy rates has been suggested. Research on the metabolism of mammalian preimplantation embryos revealed that excess lactate in the culture medium, in addition to pyruvate and glucose, can burden metabolic efficiency, as embryos naturally produce lactate during energy production. Study design, size, duration The prospective, monocentric study was designed and conducted at the Kinderwunsch Institut Schenk GmbH (Dobl, Austria). A total of 60 women with an average age of 32.5 undergoing ICSI (intracytoplasmic sperm injection) treatment were included. All of them had at least 6 mature (MII) oocytes at time of insemination. Sibling oocytes were cultured in two different culture medias and outcomes were compared. Patients undergoing preimplantation genetic testing were excluded. Participants/materials, setting, methods Mature (MII) oocytes of each patient (total 621 MII oocytes) were inseminated by ICSI and then equally split into two culture medias: standard (313 oocytes) and low lactate (308 oocytes) covered with mineral-oil overlay and incubated in time-lapse under the same conditions. A total of 428 embryos were obtained. Embryos were evaluated using morphokinetic criteria. The fertilization rate, embryo utilization rate, pregnancy rate and time to Blastocyst were analyzed. Main results and the role of chance No differences were observed in the number of fertilized oocytes (n = 211 and 217 in the standard and low lactate media, respectively). The overall embryo utilization rate was significantly different (p < 0.008) resulting in 52% in standard media and 64.3% using low lactate media. Interestingly, age stratification (18-30; 31-35; 36-41) revealed overall higher embryo utilization rates when using low lactate media in all age groups. Blastocyst utilization rate was significantly different (p < 0.007) 43,1% and 58,05% in the standard and low lactate media, respectively. Regarding the time to blastocyst, it was observed that in the standard culture media 80% of the blastocysts reached blastocyst stage on D + 5 whereas in the low lactate media 92,07% reached blastocyst stage on D + 5, which was also significantly different (p < 0.001). No differences in pregnancy and live birth rates were observed. Limitations, reasons for caution The number of patients may be seen as a study limitation. Results should be confirmed with a bigger sample size. Wider implications of the findings Low lactate media could help improve IVF outcomes due to higher embryo utilization rate leading to better cumulative pregnancy rates. Excessive lactate concentration in culture media could negatively influence embryo metabolism. Trial registration number not applicable

Chronic endometritis and the endometrial microbiota: implications for reproductive success in patients with recurrent implantation failure

BackgroundChronic endometritis (CE) is associated with poor reproductive outcomes, yet the role of endometrial microbiota in patients with recurrent implantation failure (RIF) and CE remains unclear. This study aims to characterize endometrial microbiota in RIF patients with CE and assess its implications for reproductive outcomes.MethodsIn this prospective study, we enrolled RIF patients both with and without CE. Endometrial and cervical samples were collected for 16 S rRNA gene sequencing. Microbiota composition was compared between groups using diversity indices, phylum, and genus-level analysis. Canonical correlation analysis (CCA) and Spearman’s correlation coefficients were used to assess relationships between CE, reproductive outcomes, and microbiota. Predictive functional profiling was performed to evaluate metabolic pathways associated with CE.ResultsEndometrial microbiota in CE patients exhibited greater diversity and evenness compared to non-CE patients. Principal coordinates analysis (PCoA) revealed distinct clustering between CE and non-CE groups. Linear discriminant analysis (LDA) identified Proteobacteria, Aminicenantales, and Chloroflexaceae as characteristic of CE, while Lactobacillus, Acinetobacter, Herbaspirillum, Ralstonia, Shewanela, and Micrococcaceae were associated with non-CE. CCA demonstrated associations between CE, adverse reproductive outcomes, and specific bacterial taxa. Microbial metabolic pathways significantly differed between CE and non-CE groups, with enrichment in pathways related to cofactors, vitamins, secondary metabolites, and the immune system in CE patients.ConclusionRIF patients with CE exhibit distinct endometrial microbiota compositions associated with adverse reproductive outcomes. The increased microbial diversity and altered metabolic pathways in CE suggest a potential correlation with reproductive outcomes, although further studies are necessary to elucidate the causal relationship between microbiota alterations and fertility. Modulating the endometrial microbiome may represent a novel therapeutic strategy to improve IVF outcomes in patients with CE.

Impact of Prestimulation Treatment

Polycystic ovarian syndrome (PCOS) is one of the commonest endocrine disorders encountered in women of reproductive age. It is characterised by an altered Androgen metabolism which leads to irregular menses, anovulation and hyperandrogenism (HA). It is associated with hyperinsulinaemia and obesity which aggravate the HA status. Ovulation induction and controlled ovarian stimulation pose a unique challenge in these patients. The endocrine disturbance leads to arrest of follicular growth and patients may either not respond to OI drugs are may hyper-respond leading to a risk of OHSS. The best strategy to improve outcome of IVF in PCOS patients is still under consideration. This lecture will address pre-treatment (IVF) drugs and strategies that ameliorate HA, decrease the risk of OHSS and improve cycle outcome. Selection of the safest and most effective OS protocols will be discussed. Use of segmental IVF in PCOS patients has almost eliminated OHSS- it involves using an antagonist protocol with a GnRH agonist trigger and a freeze all strategy. The pros and cons of this strategy will be discussed. We will also look at the current status of Metformin Inositols, Vit D in PCOS patients and their role in improving IVF outcome. Use of pre-treatment hormones/OCP’s and their effect on IVF outcome will be deliberated. Lifestyle change and weight reduction are important to improve the HI & HA status and have become a fundamental requirement in these young women. The challenge patients face in achieving weight reduction goals due to the inherent metabolic disturbances needs to be recognised. At the end of the lecture the delegate would be able to have a better understanding of the current status of various pre-treatment strategies used in PCOS patients undergoing IVF.

Human Menopausal Gonadotropins in Combination for Stimulation does not Improve IVF Outcomes in POSEIDON Group 4 Patients, When Compared to Recombinant Follicle Stimulating Hormone Alone: A Prospective Randomized, Non-Blinded, Controlled Pilot Trial

Human Menopausal Gonadotropins in Combination for Stimulation does not Improve IVF Outcomes in POSEIDON Group 4 Patients, When Compared to Recombinant Follicle Stimulating Hormone Alone: A Prospective Randomized, Non-Blinded, Controlled Pilot Trial

O-068 Transdermal testosterone gel (TTG) pre-treatment duration in improving ivf outcome in patients with poor prognosis (poseidon group 3 and 4): A randomised controlled trial.

Abstract Study question To compare the most effective duration of pre-treatment with transdermal testosterone gel in improving IVF outcomes using GnRH antagonist protocol in poor prognosis patients. Summary answer Pre-treatment with testosterone gel for 28 days had higher clinical pregnancy rate, more mature oocytes in lesser days and lower total dose of gonadotropins. What is known already Poor prognosis patients undergoing IVF exhibits subnormal ovarian response resulting in higher IVF cycle cancellation and lower clinical pregnancy. Amongst various adjuvants regimens use of androgen supplementation improves clinical pregnancy and probability of live birth in such patients. Androgen stimulates early stages of follicular growth, increase the number of primary, pre-antral and antral follicles. It increases FSH receptor expression in granulosa cells, in turn leading to better oocyte yield and pregnancy rate. We aim to compare the most effective duration of pre-treatment with transdermal testosterone gel in improving IVF outcomes using GnRH antagonist protocol in poor prognosis patients. Study design, size, duration A prospective, randomised controlled trial was carried out from 1st September 2021 to 31st August 2022 at a tertiary infertility centre in India. 100 poor prognosis patients POSEIDON group 3 and 4 (age 21-40 years, AFC <5 and AMH ≤1.2 ng/ml) were enrolled. They were randomized into 4 groups of 25 each by computer based program. Participants/materials, setting, methods Group 1, control received no pretreatment, Group 2, Group 3 and Group 4 received 1% TTG one actuation (12.5mg testosterone daily) for 2, 3 and 4 weeks respectively before start of ovarian stimulation. Patients received a starting dose of HP HMG dose 300 IU and dose was adjusted. Inj. Cetrorelix 0.25 mg started when lead follicle was 14 mm and Oocyte retrievals were performed 35 hours after 250 mcg r-HCG trigger. Main results and the role of chance All groups were comparable with regards to baseline FSH, AFC and AMH levels. Only one cycle was cancelled in control group because of lack of response to gonadotropins till day 5 of stimulation. Requirement of total days (9.68 Vz 8.95 days) and total dose of gonadotropins was significantly lower in group 4 as compared to control group 1. This showed that cases with pre-treatment of transdermal testosterone gel for longer duration, required less amount of gonadotropin (2826 IU Gp 1 Vz 2329 IU Gp 4) treatment (p < 0.01) and also the mean number of mature oocytes retrieved (0.80 Gp 1 Vz 3.38 Gp 4) were significantly higher (p < 0.01). Day 3 fresh embryo transfers were performed for all. Incidence of clinical pregnancy rate was 4.2%, 8%, 12% in group 1, 2, and 3 respectively which increased to 20% in group 4 (p-0.33). Miscarriage rate was 4.2% , 4%, 12% and 4% in group 1 ,2, 3 and 4 respectively (p-0.71). Ongoing pregnancy rate was 4% in group 1, while it was 7.7 %, 10.6 % and 19.2% in group 2,3 and 4 respectively (p-0.33). Limitations, reasons for caution The study was done at a single centre with small sample size, replication with more subjects and in different centers is needed. The long term effects of testosterone pretreatment requires further studies. Wider implications of the findings Pre-treatment with testosterone gel in poor prognosis patients improves response to gonadotrophin, reducing amount and days to complete stimulation and results in higher number of mature oocytes and improved clinical pregnancy rates. Transdermal testosterone treatment is advantageous as it is not first metabolized in the liver, convenient to the patient. Trial registration number CTRI/2022/03/040793

P-271 How much should you tolerate? The tolerance level of gas mixtures percentage in premixed gases

Abstract Study question Does the percentage of tolerance level of gas mixtures for premixed gas affect blastocyst formation rate? Summary answer A lower percentage of gas tolerance level can improve the blastocyst formation rate by providing a more optimal pH in the culture microenviroment. What is known already Benchtop incubators that use premixed gases are regarded as one of the most common incubators used in many IVF setups. The level of CO2 in the mixture has a direct impact on the media’s pH whereby an increase in the CO2 level will result in a lower pH and vice versa. Most media companies require a percentage of 6.0-6.5% to achieve an acceptable pH range of 7.2-7.4 for optimal culture microenvironment. However, it is often taken for granted that the CO2 and pH of the medium is within the acceptable range due to the “certificate” provided by these gas companies. Study design, size, duration This is a retrospective study looking at the average blastocyst formation rate from January 2018-December 2022 on 1665 cycles. Participants/materials, setting, methods The total MII collected and inseminated was 21,631. The mean women’s age was 37 ± 4.2 years old. All the embryos were cultured in the same laboratory conditions using BT 37 Planer (Origio) and K-Minc (Cook) incubators. From January 2018 to July 2020, all the patient’s embryos were cultured with premixed gas of 5% tolerance level (n = 940) and from Aug 2020-December 2022, all of the embryos were cultured with premixed gas of 2% tolerance level (n = 725). Main results and the role of chance It was found that the average blastocyst rate was significantly increased when the gas tolerance level was 2% (57.09%) when compared to when the gas tolerance level was 5% (52.11%) (p < 0.05). Limitations, reasons for caution Different gas companies may use different methods of measuring the gas percentage levels. Therefore, it is advisable to be cautious and impose internal QC on the gas mixture percentage on individual cylinders. Wider implications of the findings A lower gas tolerance level may provide a more optimal accuracy of gas percentage to achieve the desired pH for embryo culture and to improve IVF outcomes. Trial registration number not applicable

P-489 Uterine fluid-immune cell analysis at the time of the embryo transfer

Abstract Study question What are the subtypes of immune cells present in the upper and lower parts of the uterus at the time of frozen embryo transfer (FET)? Summary answer Our study is the first study confirming different immune cell subsets in the freshly collected uterine fluid at the time of the embryo transfer. What is known already Although endometrial receptivity is a key factor in influencing implantation in both naturally conceived and assisted reproductive technology (ART) cycles, very little is known about the endometrium milieu around the time of implantation. Previous studies have demonstrated the presence of several cytokines in the endometrium affect implantation. However, there is lacking data about the presence of immune cell subtypes within the endometrium and in the uterine cavity at the time of implantation. Study design, size, duration This study was approved by the Institutional Review Board (# 49174). The study was designed as a prospective observational cohort study between May 2021 and December 2022 at a single academic-based fertility center. All patients underwent at least one In Vitro Fertilization cycle (IVF) and have frozen embryos. Twenty-four participants were recruited for this study which was conducted during the frozen embryo transfer cycle (FET) regardless of the outcome of previous cycles. Participants/materials, setting, methods Trial transfer catheter was introduced under ultrasound guidance into lower uterine segment. Upon removal, the tip was rinsed in IMDN medium containing 10% FBS (lower). Embryo was then placed in upper uterus under ultrasound guidance. The tip of transfer catheter was rinsed in separate aliquot of the above media (upper). After centrifugation, pelleted cells were stained for the following surface markers: CD45, CD3, CD19, CD4, CD8, gamma delta TCR, CD25, CD127, CD66b, CD14, CD16, CD56. Main results and the role of chance Upon staining the pelleted cells, we were able to identify viable leukocytes from samples obtained from both, upper and lower uterus (0.1257 x 106 cells +/- SD 0.3167), (0.1230 x 106 cells +/- SD 0.1171), respectively. Among total viable cells, there was no significant difference in both the percent and number of CD45+ cells between the upper and lower uterus (9.880 +/- 6.983, 13.67 +/- 9.792, p = 0.1980) respectively. However, there was significantly higher expression of CD3 + (p = 0.006), CD19 + (p = 0.0319) and CD14 + (p = 0.0189) cells in samples collected from upper compared to lower uterus. Within all CD3+ cells, we found that gamma delta T cells (GDT) were the major population of T cells in both upper and lower uterus. In contrast, CD8+ T cells were significantly higher in the lower uterus when compared to the upper uterus (p = 0.009). There was no statistically significant difference in the expression of CD4+ T cells, T regulatory cells, NK cells (CD56+), neutrophils (CD66b+) and monocytes (CD14+) between upper and lower uterus. Limitations, reasons for caution Limitations of our study include number of participants as well as the number of parameters analyzed by flow cytometry. To maximize information from each sample, multicolor flow cytometry can give critical insight into immune cell composition of the uterine milieu and the difference in expression between upper and lower uterus. Wider implications of the findings We believe the immune milieu at the time of embryo transfer will affect implantation. Understanding the composition of immune cell will guide further research in identifying optimal immune milieus that favor implantation. Comprehensive analysis of endometrium is expected to lead to new diagnostic and therapeutic approaches to improve IVF outcomes. Trial registration number not applicable

O-295 Dual trigger does not improve reproductive outcomes in advanced maternal age women

Abstract Study question Could dual trigger increase oocyte yield and maturation rate in women of advanced maternal age (≥ 40) undergoing IVF? Summary answer Dual trigger does not result in higher oocyte yield or maturation rates compared to hCG monotrigger What is known already Advanced maternal age is related to poor ovarian response (POR) and remains a major therapeutic challenge in routine IVF practice, because of the association with low live birth rates and high cancellation rates. hCG is used at the end of controlled ovarian hyperstimulation as a surrogate LH surge to induce final oocyte maturation. Recently, the co-administration of GnRH agonist and hCG for final oocyte maturation (dual trigger) has been suggested to improve IVF outcome (by improving oocyte quantity and quality) in normal responders, while evidence in poor responders remains controversial. Study design, size, duration This is a retrospective cohort study including patients attending a private IVF clinic from 1st January 2018 until 1st June 2022. Participants/materials, setting, methods All women who underwent IVF/ICSI in antagonist protocol in our center were included. Patients may have undergone triggering of the final oocyte maturation either with 250mcg of rhCG or 0.2 mg of GnRH agonist. Mature oocytes were inseminated using ICSI. Main results and the role of chance In total, 2242 patients were included, 454 (20.2%) in the rhCG group and 1788 (79.8%) in the dual trigger group. There was no significant difference in female age [41.3 (1.12) vs 41.3 (1.19), p value 0.94]. Total stimulation units and duration were also comparable between groups. The number of oocytes and MII oocytes did not differ significantly between rhCG and dual trigger group [5.8 (4.1) vs 6.3 (4.7) and 4.7 (3.3) vs 4.9 (3.4), p = 0.15 and 0.49, respectively]. Maturation rates were similar 81.7% (22.4) and 79.8% (23.5), p = 0.15, as well as fertilization rates (defined as the number of oocytes fertilized divided by the total number of cumulus-oocyte complexes recruited) [57% (29.7) vs 58.8% (29.4), p value=0.2]. Embryo utilization rates (defined as the total number of embryos transferred and cryopreserved divided by the number of oocytes fertilized) were comparable between the two arms: 69.7% (34.2) vs 70% (33.9), p value =0.22. Multivariate Poisson regression analysis adjusting for relevant confounders (AMH, total stimulation units) showed that the type of triggering strategy (dual trigger vs rhCG) was not associated with either the number of MII oocytes (coefficient 0.2, p value=0.24) or maturation rates (coefficient -1.8, p value 0.12). Limitations, reasons for caution The main limitation is the retrospective design of our study, with an inherent risk of bias. Wider implications of the findings To the best of our knowledge, this is the largest study evaluating dual trigger strategy in advanced maternal age women. Our data demonstrate that dual trigger cannot improve the outcome of low prognosis women and should not be used as a panacea for all IVF patients. Trial registration number NA

Does the Incorporation of Dual Trigger Improve IVF Outcome When Compared to HCG Trigger in IVF / ICSI Cycles?

Does the Incorporation of Dual Trigger Improve IVF Outcome When Compared to HCG Trigger in IVF / ICSI Cycles?

Subtle changes in perivascular endometrial mesenchymal stem cells after local endometrial injury in recurrent implantation failure

Improvements in reproductive techniques have resulted in the live birth rates from IVF procedures increasing from 5% to approximately 30% in recent decades but has plateaued since. Emerging preclinical and clinical data implicates endometrial receptivity deficiencies in patients with recurrent implantation failure (RIF) as the predominant factor hindering successful implantation. Mechanisms on how local endometrial injury (LEI) improves implantation rates in patients with RIF are currently unknown. We hypothesized that LEI may influence perivascular endometrial mesenchymal stem/progenitor cells (eMSCs) which are thought to regenerate the stromal vascular component of the functional layer every month. Here, we assessed the effect of LEI on the proportion and function of eMSCs present in consecutive LEI biopsies. Consecutive paired mid-luteal phase endometrial biopsies obtained from patients with RIF were digested to single cells and the proportion of SUSD2-expressing cells determined. Growth kinetics and decidualization were compared between the consecutive LEI samples. A mid-luteal LEI altered the decidualization capacity of SUSD2+ eMSCs in women with RIF, but not their proportion or clonogenicity. With the potential of LEI to improve IVF outcomes in women with RIF, additional investigations are needed to understand the impact of the altered decidualization response in eMSCs.

Therapeutic intervention based on gene sequencing analysis of microbial 16S ribosomal RNA of the intrauterine microbiome improves pregnancy outcomes in IVF patients: a prospective cohort study.

A Lactobacillus-dominated microbiota in the endometrium was reported to be associated with favorable reproductive outcomes. We investigated in this study whether 16S ribosomal RNA (rRNA) gene sequencing analysis of the uterine microbiome improves pregnancy outcomes. This prospective cohort study recruited a total of 195 women with recurrent implantation failure (RIF) between March 2019 and April 2021 in our fertility center. Analysis of the endometrial microbiota by 16S rRNA gene sequencing was suggested for all patients who had three or more failed embryo transfers (ETs). One hundred and thirty-one patients underwent microbial 16S rRNA gene sequencing (study group) before additional transfers, while 64 patients proceeded to ET without that analysis (control group). The primary outcome was to compare the cumulative clinical pregnancy rate of two additional ETs. An endometrial microbiota considered abnormal was detected in 30 patients (22.9%). All but one of these 30 patients received antibiotics according to the bacterial genus detected in their sample, followed by treatment with probiotics. As a result, the cumulative clinical pregnancy rate (study group: 64.5% vs. control group: 33.3%, p = 0.005) and the ongoing pregnancy rate (study group: 48.9% vs. control group: 32.8%, p = 0.028) were significantly increased in the study group compared to the control group. Personalized treatment recommendations based on the microbial 16S rRNA gene sequencing of the uterine microbiota can improve IVF outcomes of patients with RIF. The University Hospital Medical Information Network (UMIN) Clinical Trial Registry: UMIN000036050 (date of registration: March 1, 2019).

Pituitary suppression with GnRH agonists before ART may be insufficient to treat women with severe adenomyosis

Research questionDoes aromatase inhibitor improve IVF outcomes by reducing local oestrogen production in patients with adenomyosis undergoing long-term gonadotrophin-releasing hormone agonist (GnRHa) treatment? DesignFour patients with severe adenomyosis who failed to improve after long-term treatment (≥3 months) with depot GnRHa received treatment with an aromatase inhibitor for 21 days. Blood oestradiol concentrations were monitored after GnRHa treatment both before and after treatment with an aromatase inhibitor. Women received a transfer of IVF autologous or donor oocytes. Pregnancy and ongoing pregnancy rates were the primary outcomes. Blood oestradiol concentration after treatment with an aromatase inhibitor was a secondary outcome. ResultsPatients with severe adenomyosis presented with hyperestrogenism due to local production from the lesions even after long-term treatment with GnRHa. Treatment with an aromatase inhibitor reduced hyperestrogenism and improved clinical outcomes in adenomyosis patients who have experienced previous embryo transfer failures. ConclusionWomen with severe adenomyosis would benefit from letrozole or a combination of GnRHa plus letrozole before receipt of treatment with assisted reproductive technology. For women with severe adenomyosis, GnRHa treatment alone may be insufficient to suppress oestrogen production by adenomyotic lesions. Thus, it should be mandatory to test for oestradiol concentrations in patients with severe adenomyosis who have received long-term GnRHa treatment. Also, GnRHa may not always be the sole strategy for medical management of adenomyotic lesions. Letrozole is safe and can improve IVF outcomes for patients with adenomyosis.

Adjustment of progesterone administration after endometrial transcriptomic analysis does not improve reproductive outcomes in women with adenomyosis

Research questionDo patients with adenomyosis present a dysregulated endometrial receptivity that can be reversed with personalized embryo transfer (PET) by transcriptomic-based progesterone adjustment, improving IVF outcomes? DesignA multicentre, retrospective, cohort study that transcriptomically analysed the endometrial receptivity of the endometrium in patients with adenomyosis (n = 81) and healthy women (n = 231). Subsequently, implantation, biochemical and clinical miscarriage, and live birth rates between adenomyosis patients with one previous implantation failure using donor oocytes who received (n = 59) or did not receive (n = 66) PET based on endometrial receptivity, were observed to evaluate if adjusted progesterone improves reproductive outcomes of adenomyosis patients. ResultsPatients with adenomyosis significantly presented an altered endometrial receptivity (non-receptive) compared with healthy patients (53.1% versus 37.2%, P = 0.0179), elevating the risk of adenomyosis patients having a non-receptive endometrium 42.59% higher (95% CI 41.50 to 44.45). No significant differences were found in implantation (62.7% versus 78.8%, P = 0.0514), biochemical (13.5% versus 3.9%, P = 0.1223) and clinical (10.8% versus 15.4%, P = 0.7543) miscarriage, or live birth rates (75.7% versus 80.8%, P = 0.6066), in patients with PET compared with those without PET. ConclusionsWomen with adenomyosis presented an altered expression of genes involved in decidualization, and a higher rate of non-receptive endometrial statuses than controls. Although progesterone is indispensable for implantation, adjusting progesterone before PET, using endometrial transcriptomic signatures, does not improve IVF outcomes in patients with adenomyosis. Other molecular mechanisms beyond progesterone regulation may be involved in implantation failure.

Efficacy of Calcium Chloride Dihydrate Supplementation at ICSI on Patient Outcomes

Background: ICSI typically results in fertilisation rates of >65% however patients may still have low or no fertilisation despite the oocyte and sperm quality appearing normal. After entry of the sperm into the oocyte, a cascade of reactions facilitates calcium release and subsequent oocyte activation. In cases of poor or failed fertilisation it is possible that this cascade fails and therefore different methods of artificial oocyte activation are used to try and induce calcium release. Aim: To determine if calcium chloride dihydrate supplementation in the PVP (5mM) at the time of ICSI (ICSI-Ca) improves fertilisation, utilisation and clinical pregnancy rates compared to ICSI alone, particularly in patients with a history of low fertilisation (<50%). Methods: Retrospective study between 2016-2021 at Monash IVF Victoria on a paired cohort of patients (n=178 patients) where an ICSI cycle was analysed coupled with the subsequent ICSI-Ca cycle. The paired cohort was further subdivided into a low-fertilisation cohort (<50% fertilisation on previous cycles: n=66 patients) compared to the remaining patients with normal-fertilisation ([Formula: see text] 50%; n=122). Exclusion criteria included donor cycles, PGT patients, surgical sperm retrieval, women [Formula: see text] 45 years old, patients with >6 cycles and [Formula: see text] 5 inseminated oocytes. Results: Calcium supplementation increased both fertilisation (28.8% ICSI vs 49.7% ICSI-Ca, p<0.0001) and clinical pregnancy rate (4.9% ICSI vs 25.0% ICSI-Ca: p<0.05) in the low-fertilisation cohort but not in the normal-fertilisation cohort. Interestingly, utilisation rate increased in the normal-fertilisation cohort (32.6% ICSI vs ICSI-Ca: 44.9%, p<0.01) but not in the low-fertilisation cohort, although the number of extra embryos utilised per patient after ICSI-Ca increased in both groups. Conclusion: Calcium supplementation appears not to be a detrimental addition to ICSI and may improve IVF outcomes, particularly for patients with a history of low fertilisation. Further investigations including prospective case matched studies or RCT are required to confirm these findings.