Assisted Reproductive Cycles Research Articles

Magnetic-Activated Cell Sorting as a Method to Improve Necrozoospermia-Related Asthenozoospermic Samples.

According to some statistics, absolute asthenozoospermia affects every 1 in 5000 men. Although this incidence rate does not appear to be too high, it is extremely important to address the phenomenon because it can drastically reduce the chances of pregnancy, even with assisted reproduction. The biggest problem with absolute asthenozoospermia is that it is difficult to distinguish between live and dead sperm cells, and fertilization with non-viable spermatozoa may contribute to the failure of an assisted reproduction cycle. Nowadays, DNA fragmentation (DF) is a crucial parameter of semen analysis, and in this paper, we provide evidence of the correlation between DF and vitality. For this purpose, the main semen parameters were investigated by a CASA system (concentration, motility, progressive motility, vitality and DF). In the necrozoospermic group (vitality < 58%), all the measured parameters showed significant differences compared to normal vitality. Concentration (30.1 M mL−1 vs. 13.6 M mL−1), motility (31.9% vs. 18.3%), and progressive motility (24.3% vs. 12.7%) were significantly decreased, while DF was significantly increased (17.4% vs. 23.7%). Based on the connection between vitality decrement and DF increment, DF lowering methods, such as magnetic-activated cell sorting, have been hypothesized as novel methods for the elimination of dead spermatozoa.

Outcomes of Empirical Treatment With Intravenous Immunoglobulin G Combined With Low-Dose Aspirin in Women With Unexplained Recurrent Pregnancy Loss.

BackgroundTo assess the clinical efficacy of intravenous immunoglobulin G (IVIG) administration combined with low-dose aspirin in women with unexplained recurrent pregnancy loss (RPL).MethodsWe retrospectively analyzed the medical records of patients who had been diagnosed with unexplained RPL and treated with IVIG and low-dose aspirin between January 2000 and March 2020 at Asan Medical Center. We analyzed pregnancy outcomes and their association with the percentage of natural killer (NK) cells.ResultsThe study analyzed a total of 93 patients and 113 natural and assisted reproductive technology pregnancy cycles. The live birth rate per cycle was 73.5% (83/113), and the term delivery rate was 86.7% (72/83). The live birth rate was high regardless of the type of RPL, method of pregnancy, timing of IVIG treatment, and presence or absence of autoantibodies. In addition, the live birth rate was significantly higher in patients who received IVIG more than once, compared with patients who received IVIG only once (77.8% vs. 42.9%, P = 0.006). There was no significant association between the NK cell counts and live birth rate (65.5% in the group with NK cell < 12%, and 69.7% in that with NK cell ≥ 12%, P = 0.725). Among all patients, 87.6% had no complications, and there were no congenital malformation among newborn babies.ConclusionIVIG combined with low-dose aspirin treatment showed favorable pregnancy outcomes regardless of the patient’s NK cell counts (%).

Influence of Women´s Residence Region on Assisted Reproduction Treatments - Experience of a Tertiary Center in Northern Portugal.

ObjectiveData on the possible influence of women´s region of residence, within the same country, on the outcomes of medically assisted reproduction cycles are scarce. This study aims to assess the impact of the women's region of residence on the results of in-vitro fertilization cycles.MethodsWe evaluated in-vitro fertilization cycles between 2010 and 2017, performed in a northern Portugal assisted reproduction center. We defined two groups: Douro Litoral (group 1; n=783), and Trás-os-Montes and Alto Douro (group 2; n=178). We analyzed demographics and cycle-related variables, and we calculated the rates for embryo transfer cycles. We used the Mann-Whitney and Chi-square tests and p<0.05 was considered statistically significant.ResultsWe included 961 cycles. The region of residence had no effect on the following variables: women´s age; body mass index; or duration of infertility (p>0.05). Group 2 had a statistical significant lower number of previous cycles than group 1 (1.3±0.5 and 1.5±0.7; p=0.005). In the sub-analysis of cycles with embryo transfer (n=781), group 1 obtained had rates of normal fertilization (62.5% vs. 57.5%; p=0.04), miscarriage rate (30.0 vs. 10.9%; p=0.007), and lower implantation rates compared to group 2 (33.3% vs. 50.0%; p<0.001).ConclusionsWomen from the region of Trás-os-Montes e Alto Douro had a lower number of previous cycles, compared to those from the Douro Litoral, despite the absence of statistical significant differences in terms of age or infertility duration. These findings reinforce the need to contemplate the sociodemographic and socioeconomic variables in this context.

Intramuscular Progesterone Optimizes Live Birth From Programmed Frozen Embryo Transfer: A Randomized Clinical Trial

(Abstracted from Fertil Steril 2021;116:633–643) Approximately one third of assisted reproductive cycles in the United States are classified as frozen embryo transfer (FET). Given the widespread utilization of FET in the treatment of infertility, identifying endometrial preparation protocols that optimize live birth is critical.

Extensive analysis of mitochondrial DNA quantity and sequence variation in human cumulus cells and assisted reproduction outcomes.

Are relative mitochondrial DNA (mtDNA) content and mitochondrial genome (mtGenome) variants in human cumulus cells (CCs) associated with oocyte reproductive potential and assisted reproductive technology (ART) outcomes? Neither the CC mtDNA quantity nor the presence of specific mtDNA genetic variants was associated with ART outcomes, although associations with patient body mass index (BMI) were detected, and the total number of oocytes retrieved differed between major mitochondrial haplogroups. CCs fulfil a vital role in the support of oocyte developmental competence. As with other cell types, appropriate cellular function is likely to rely upon adequate energy production, which in turn depends on the quantity and genetic competence of the mitochondria. mtDNA mutations can be inherited or they can accumulate in somatic cells over time, potentially contributing to aging. Such mutations may be homoplasmic (affecting all mtDNA in a cell) or they may display varying levels of heteroplasmy (affecting a proportion of the mtDNA). Currently, little is known concerning variation in CC mitochondrial genetics and how this might influence the reproductive potential of the associated oocyte. This was a prospective observational study involving human CCs collected with 541 oocytes from 177 IVF patients. mtDNA quantity was measured in all the samples with a validated quantitative PCR method and the entire mtGenome was sequenced in a subset of 138 samples using a high-depth massively parallel sequencing approach. Associations between relative mtDNA quantity and mtGenome variants in CCs and patient age, BMI (kg/m2), infertility diagnosis and ART outcomes were investigated. Massively parallel sequencing permitted not only the accurate detection of mutations but also the precise quantification of levels of mutations in cases of heteroplasmy. Sequence variants in the mtDNA were evaluated using Mitomaster and HmtVar to predict their potential impact. The relative mtDNA CC content was significantly associated with BMI. No significant associations were observed between CC mtDNA quantity and patient age, female infertility diagnosis or any ART outcome variable. mtGenome sequencing revealed 4181 genetic variants with respect to a reference genome. The COXI locus contained the least number of coding sequence variants, whereas ATPase8 had the most. The number of variants predicted to affect the ATP production differed significantly between mitochondrial macrohaplogroups. The total number of retrieved oocytes was different between the H-V and J-T as well as the U-K and J-T macrohaplogroups. There was a non-significant increase in mtDNA levels in CCs with heteroplasmic mitochondrial mutations. N/A. Although a large number of samples were analysed in this study, it was not possible to analyse all the CCs from every patient. Also, the results obtained with respect to specific clinical outcomes and macrohaplogroups should be interpreted with caution due to the smaller sample sizes when subdividing the dataset. These findings suggest that the analysis of mtDNA in CCs is unlikely to provide an advantage in terms of improved embryo selection during assisted reproduction cycles. Nonetheless, our data raise interesting biological questions, particularly regarding the interplay of metabolism and BMI and the association of mtDNA haplogroup with oocyte yield in ovarian stimulation cycles. This study was funded by National Institutes of Health grant 5R01HD092550-02. D.J.N. and C.R. co-hold patent US20150346100A1 and D.J.N. holds US20170039415A1, both for metabolic imaging methods. D.W. receives support from the NIHR Oxford Biomedical Research Centre. The remaining authors have no conflicts of interest to declare.

The Protective Effect of Sulforaphane against Oxidative Stress through Activation of NRF2/ARE Pathway in Human Granulosa Cells.

Objective Sulforaphane (SFN) is a natural free radical scavenger that can reduce oxidative stress (OS) through mediating nuclear factor (erythroid-derived 2)-like 2 (NF-E2-related factor 2 or NRF2)/antioxidant response element (ARE) signaling pathway and the downstream antioxidant enzymes. Here, we intended to study the role of SFN in OS- induced human granulosa cells (GCs) by investigating the intracellular levels of reactive oxygen species (ROS), cell death, and NRF2-ARE pathway. Materials and Methods This experimental study was conducted on GCs of 12 healthy women who had normal menstrual cycles with no history of polycystic ovary syndrome (PCOS), endometriosis, menstrual disorders, hyperprolactinemia, or hormonal therapy. After isolation of GCs, the MTT assay was performed to explore GCs viability after treatment with SFN in the presence or absence of H2O2. Flow cytometry was utilized to determine the intracellular ROS production and the apoptosis rate. Evaluation of the mRNA and protein expression levels of NRF2 and phase II enzymes including superoxide dismutase (SOD) and catalase (CAT) was performed by quantitative real-time polymerase chain reaction (PCR) and western blotting. Finally, the data were analyzed by SPSS software using One-way ANOVA and the suitable post-hoc test. Significance level was considered as P<0.05. Results Pretreatment of GCs with SFN attenuated intracellular ROS production and apoptosis rate in the H2O2-exposed cells. Moreover, SFN treatment increased the mRNA expression level of NRF2, SOD, and CAT. Higher expression of NRF2 and SOD was also observed at the protein level. ConclusionOur study demonstrated that SFN protects human GCs against H2O2induced-OS by reducing the intracellular ROS production and the following apoptosis through a mechanism by which NRF2 increases the antioxidant enzymes such as SOD and CAT. This result may have a potential application in assisted reproduction cycles by improving the quality of GCs and the embedded oocyte, especially in PCOS patients.

Vitamin D receptor gene polymorphisms and the effectiveness of assisted reproductive technology

Introduction. Vitamin D administration is recommended for women with vitamin D insufficiency, who are planning to undergo assisted reproductive treatment (ART). The action of the active form of vitamin D is mediated through its vitamin D receptor (VDR). The presence of VDR in the cells and tissues of the female reproductive system suggests that vitamin D plays an important role in human reproduction. However, the effect of VDR gene polymorphisms on human reproduction has not been adequately studied.Aim. To assess the effect of VDR gene polymorphisms on the folliculogenesis, embryogenesis and clinical outcomes of assisted reproduction programs.Materials and methods. The cross-sectional study included 300 patients without complications during the assisted reproduction cycle. The number of follicles, oocyte-cumulus complexes, mature and immature oocytes, the presence of oocyte dysmorphisms, number of zygotes, fertilization rate, and the number of obtained blastocysts were evaluated. The determination of three polymorphisms of the VDR gene (FokI – rs2228570, BsmI – rs1544410, TaqI – rs731236) was performed by real-time polymerase chain reaction.Results. The incidence of perivitelline space pathology of oocytes in FokI A/A genotype patients was 5.7%, in A/G genotype patients – 14.7%, in G/G genotype patients – 16.3% (p = 0.041 when comparing A/A genotype with A/G + G/G genotypes). Depending on the genotype, the pregnancy rate ranged from 38.5 to 52.8%, but no statistically significant differences were found.Conclusion. It was shown that the perivitelline space pathology of oocytes in the FokI A/G genotype patients is 2.6 times higher, and in the G/G genotype patients – 2.9 times higher than in the A/A genotype patients. However, there were no differences in pregnancy rate among patients with different genotypes of FokI, BsmI and TaqI gene polymorphisms of VDR.

Effects of multiple doses of gonadotropin-releasing hormone agonist on the luteal-phase support in assisted reproductive cycles: A clinical trial study.

BackgroundThe effect of adding gonadotropin-releasing hormone (GnRH) agonist on the luteal phase support in assisted reproductive technique (ART) cycles is controversial.ObjectiveTo determine the effects of adding multiple doses of GnRH agonist to the routine luteal phase support on ART cycle outcomes.Materials and MethodsThis clinical trial study included 200 participants who underwent the antagonist protocol at the Research and Clinical Center for Infertility, Yazd, Iran, between January and March 2020. Of the 200, 168 cases who met the inclusion criteria were equally divided into two groups – the case and the control groups. Both groups received progesterone in the luteal phase, following which the case group received GnRH agonist subcutaneously (0/1 mg triptorelin) zero, three, and six days after the fresh embryo transfer, while the control group did not receive anything. Finally, chemical and clinical pregnancy rates, number of mature oocytes, fertilization rate, total dose of gonadotropin, and the estradiol level were determined.ResultsThe baseline characteristics were similar in both groups. No significant difference was observed between embryo transfer cycles. Clinical results showed that differences between the fertilization rate, chemical and clinical pregnancies were not significant.Conclusion The results showed that receiving multiple doses of GnRH agonist in the luteal phase of ART cycles neither improves embryo implantation nor the pregnancy rates; therefore, further studies are required.

Sperm DNA fragmentation index and cumulative live birth rate in a cohort of 2,713 couples undergoing assisted reproduction treatment

To study how the choice of the first assisted reproductive technology treatment type affects the cumulative live birth rate (CLBR) in couples with high sperm DNA fragmentation index (DFI). Longitudinal cohort study. University-affiliated fertility clinic. A total of 2,713 infertile couples who underwent assisted reproductive technology treatment between 2007 and 2017 were included in the study. All invitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatments (up to three fresh treatments and all associated frozen-thawed embryo transfers) offered to the couples by the public health care system were included, in total 5,422 cycles. None. The primary outcome was the CLBR. The secondary outcomes were the fertilization rate and the miscarriage rate. The IVF and ICSI groups were defined according to the method applied in the first treatment cycle. In the IVF group, the CLBR values were higher for couples with normal DFI compared with those for couples with high DFI (≥20%) (48.1% vs. 41.6% for conservative CLBR estimate and 55.6% vs. 51.4% for optimal CLBR estimate after adjustment for female age, respectively). No DFI-dependent difference was seen in the ICSI group. Our results demonstrated that a high DFI predicts a statistically significantly lower CLBR if IVF and not ICSI is applied in the first cycle of assisted reproduction.

P–382 Association of extended culture to blastocyst and gestational trophoblastic disease risk following IVF/ICSI assisted reproduction cycles: An analysis of large UK National database

Abstract Study question Is there any association between stage of embryo at transfer based on extended in vitro culture and gestational trophoblastic disease risk during assisted reproduction? Summary answer No significant association between stages of embryo transfer from zygote stage to blastocyst stage was found after analysing 540376 cycles of IVF and ICSI. What is known already Gestational trophoblastic disease (GTD), commonly referred to as molar pregnancy, results from abnormal proliferation of the trophoblast with a reported incidence of ∼1 in 700 in the UK. Despite technological advances such as ICSI, PGT and selection of normally fertilised (2PN) embryos, there are reported cases of GTD following assisted reproduction. Blastocyst transfer is associated with higher pregnancy and live birth rates but evidence is lacking whether extended embryo culture to blastocyst stage influences implantation of an abnormal embryo or abnormal trophoblastic proliferation leading to GTD. Study design, size, duration A retrospective study was carried out using Human Fertilisation and Embryology Authority (HFEA) anonymised register data from 1999 to 2016. HFEA holds the longest running register for fertility treatment data in the world and is the national database for fertility treatment data in UK. A total of 540376 fresh IVF or ICSI assisted reproduction cycles were analysed. Participants/materials, setting, methods There were 1033588 treatment cycles during the study period but only 540376 cycles met the inclusion criteria of fresh IVF or ICSI. Cycles with incomplete data, frozen embryo transfers, donor treatment or surrogacy were excluded. A subgroup analysis of those with primary subfertility was performed after excluding subjects with secondary infertility in order to exclude an effect of a previous molar pregnancy. Multivariate regression analysis was used to adjust for possible confounders. Main results and the role of chance 78 molar pregnancies were reported in the original sample giving a prevalence of 4/10000 live births (78/228461), much lower than the prevalence given with natural pregnancies. Prevalence of molar pregnancy amongst the study population after meeting exclusion criteria was 4 /10000 livebirths (53/156683). Incidence of molar pregnancy was not statistically different between treatment type (0.0001 vs 0.00009). Significantly higher incidence of GTD was seen in the 40 to 42 age category compared to 18–34 category(OR 1.86(95% CI 8.7–3.75)),in par with known higher GTD risk in women above 40 in the general population. Of interesting note, although the incidence of molar pregnancy was significantly lower in women undergoing assisted reproduction increased risk with advancing age is not totally eliminated with treatment. There was no significant association between the occurrence of molar pregnancy with the type and cause for infertility and number of embryos transferred. Crude (1.06 (95% CI 0.852–1.31)) and adjusted odds ratios (1.07 (95% CI (0.857–1.32)) did not show any association between day of embryo transfer and molar pregnancy even after adjusting for age and excluding secondary infertility. Selection of blastocyst stage embryo after extended culture did not alter the likelihood of having a GTD compared to cleavage stage embryo. Limitations, reasons for caution The retrospective analysis of anonymised HFEA data limited adjustments for confounders such as smoking, previous history of GTD, ethnicity etc that predispose to GTD. Caution needs to be exercised for under-reporting of GTD to HFEA and lack of information on type of GTD identified. Wider implications of the findings: Though GTD cannot be prevented by IVF/ICSI, the incidence is significantly low and extended culture is not associated with higher risk of abnormal trophoblastic proliferation or GTD occurrence with IVF/ ICSI treatment. These findings would aid informed implications counselling and reassurance of patients during assisted reproduction treatments. Trial registration number Not applicable

P–279 Effect of Ca2+ ionophore A23187 (calcymicin) on fertilization rates, embryo development and pregnancy in human assisted reproduction cycles

Abstract Study question Does Calcymicin improve reproductive outcomes of ICSI cycles in cases of fertilization failure and/or embryo blockage indications? Summary answer The application of the Calcymicin after ICSI improves reproductive outcomes, especially in cases with clinical indication of fertilization failure. What is known already According to the bibliography, deficiencies in the oocyte activation process frequently lead to failed ICSI cycles, and these can be corrected by increasing initial levels of calcium (Ca2+) in the oocyte using assisted oocyte activation techniques (AOA), such as the use of Ca2+ ionophores. Ca2+ Ionophores have been shown to trigger an initial Ca2+ spike in the ooplasm that activates Ca2+/Calmodulin dependent protein kinase II, which initiates the cascade of cellular events leading to oocyte activation. Previous results suggest that Ca2+ ionophore treatment can give live offspring after failed ICSI cycles. Study design, size, duration 270 oocytes collected from 17 patients who presented cycles with low fertilization rates and/or embryo blockage or poor quality embryos (according to ASEBIR’s embryo classification criteria) were retrospectively analyzed. Oocytes were divided into two groups, a control group that underwent conventional IVF/ICSI and another group that underwent an ICSI cycle with AOA. Study groups were defined according to clinical indications and subgroups according to AOA or control. All data were collected from 2017 until 2020. Participants/materials, setting, methods Among the 270 oocytes of the study sample, 142 belonged to the control group and 128 belonged to the AOA group. The AOA group oocytes were activated for 15 minutes immediately after ICSI using a prepared solution containing the Ca2+ ionophore A23187, CultActive© (Gynemed, Germany). Fertilization rate and type, blastocyst formation rate, blastocyst quality, embryo kinetics, and pregnancy rates were analyzed, all of them were compared to FIV/ICSI cycles without oocyte activation (control group). Main results and the role of chance In the analyses of the whole sample of oocytes, the AOA treatment gave a fertilization rate of 72.5%, which was significantly higher compared to 53.8% of the control cycles (p = 0.002). Good quality blastocysts and pregnancy rates were also significantly higher than the control (p = 0.01). In the group with an indication of fertilization failure, a significantly higher fertilization rate was recorded compared to the control (65% and 33%, respectively). A higher rate of abnormal embryos with three pronuclei was also found compared to the control (p &amp;lt; 0.001). There were no significant differences in blastocyst formation rates, quality, or embryo kinetics (p &amp;gt; 0.05). In the group with an indication of embryo blockage/poor embryo quality, a significantly higher rate of good quality blastocysts and lower blastulation time were recorded compared to the control (p &amp;lt; 0.05). Limitations, reasons for caution The safety of the AOA technique with Ca2+ ionophore has not been fully demonstrated. In our study, none of the newborns had malformations, and gestational weeks and birth weights were normal. However, further studies on the safety of this technique are needed to implement it routinely in human reproduction clinics. Wider implications of the findings: According to these findings, an increase in the initial levels of calcium in the oocyte through the application of the Ca2+ ionophore A23187 after ICSI improves the results of failed assisted reproduction cycles, especially in the case of those diagnosed with fertility failure, which is a clear indication for AOA. Trial registration number Not applicable

P–289 Progesterone receptor is not downregulated in endometrial epithelial compartment during embryo receptivity phase in assisted reproductive cycles

Abstract Study question Is progesterone receptor (PGR) downregulation disrupted within endometrial epithelial compartment, during embryo receptivity phase in assisted reproductive technology (ART) cycles? Summary answer PGR is not downregulated in endometrial epithelial cells from ART cycles during embryo receptivity phase. What is known already Progesterone (P4) promotes the downregulation of its own progesterone receptor (PGR). During the mid-luteal phase, PGR is downregulated in endometrial epithelial cells (EEC), a critical process for embryo implantation. Embryos are unable to attach to the maternal surface when PGR expression is sustained in EEC. Non-physiologic ovarian steroid produced or employed in ART cycles may alter endometrial development compromising its receptivity. Scarce information is available whether PGR is downregulated in EEC from ARTs including ovarian stimulation for in vitro fertilization (IVF) cycles or hormonal endometrial preparation for frozen thawed embryo transfer (HEP-FET). Study design, size, duration Cross sectional study including endometrial samples from fertile women during natural cycle (FNC, n = 23), from infertile women submitted to IVF (n = 19) and from infertile women who underwent mock HEP-FET (n = 35). Samples were obtained between 2018–2019. Sample size was calculated considering a power of 90%, alpha error=0.05, an expected PGR expression of 2 and 0.5 in ART and FNC groups, respectively, having a standard deviation=0.9. At least 9 patients would be necessary in each group. Participants/materials, setting, methods Endometrial samples were obtained during mid-luteal phase scheduled 7 days after ovulation in FNC, 5 days after oocyte retrieval in IVF without embryo transfer or 5 days after P4 supplementation in HEP-FET. Immunohistochemistry was employed to quantify PGR using histologic score (Hscore). PGR mRNA levels were determined by qRT-PCR from EEC dissected by laser capture microdissection. Anova test was used for comparing means of Hscore and mRNA among groups. Statistical significance was established as P &amp;lt; 0.05. Main results and the role of chance No statistical differences were found in demographic characteristics including age, body mass index or endometrial thickness. The PGR expression was reduced in FNC compared to IVF and HP-FET endometria (0.6 ± 0.1, 1.9 ± 0.9 and 2.2 ± 0.9 respectively; P &amp;lt; 0.0001). The PGR mRNA levels from ECC dissected by laser capture microdissection were higher in IVF and HP-ET cycles compared to FNC (10.6 ± 3.1, 13.6 ± 2.3 and 0.8 ± 0.1 respectively; P &amp;lt; 0.0001) corroborating the elevated PGR Hscore in EEC from ART cycles. Limitations, reasons for caution This is a descriptive study reporting failure of PGR downregulation in endometria from ART cycles with vaginal P4 supplementation during the luteal-phase. Whether interference or resistance to P4 signal is the mechanism involved in the failure of PGR down regulation in ART cycles needs to be determined Wider implications of the findings: PGR downregulation within EEC was shown in FNC. The retained PGR expression detected in most ART cycles may interfere with embryo implantation and might explain the restricted pregnancy success. Future studies might reveal whether PGR evaluation in EEC can predict embryo implantation. Trial registration number Not Aplicable

O-156 Fertilization rate as a novel indicator for cumulative live birth rate: multicenter retrospective cohort study of 9,394 complete IVF cycles

Abstract Study question Does fertilization rate (FR) affect cumulative success rates in assisted reproduction cycles? Summary answer These data indicate a positive association between FR with CLBR suggesting the predictive clinical relevance of this parameter and its adoption as Key Performance Indicator(KPI). What is known already Numerous studies have aimed at characterizing outcome predictors. Maternal age is historically and correctly recognized as the single most important factor impacting on the clinical outcome of ART. More recently ovarian response has also gained interest in this respect. However, the quest for novel, more comprehensive predictive factors is not over; new relevant evidence is starting to emerge. FR is a noteworthy parameter because expressing a fundamental aspect of both oocyte and sperm developmental competence. In fact it has been adopted as a key performance indicator of the IVF laboratory, to assess laboratory, operator, and gamete competence. Study design, size, duration Reported data concern a retrospective cohort study carried out between 2015 to 2017 involving 7,968 couples undergoing 9,394 complete ICSI cycles, i.e. whose all embryos were transferred or disposed.All women aged between 18-42 years were included.We excluded from analysis: surgical sperm retrieval cases, cycles resulting in neither fresh or frozen–thawed embryo transfers,cycles in which live birth were not achieved, but with remaining cryopreserved embryos,cycles of PGT, cycle with fertilization failure and standard IVF cycles. Participants/materials, setting, methods The cohort was groupped according to fertilization rate intervals based on recommendations of the Vienna Consensus (&amp;lt;65% - Group 1; 65%-80% - Group 2; &amp;gt;80% - Group 3). Harnessing the large size of the original dataset, further cycle stratifications were carried out based on female age (&amp;lt;34, 35-38, 39-42 years) and number of oocytes retrieved (5-7, 8-10, &amp;gt;10 oocytes). Main results and the role of chance No significant difference in female age was observed between fertilization rate groups (p = 0.640). CLBR was progressively higher in relation fertilization rate in Groups 1, 2 and 3 (20.1%, 34.7%, 41.3%, P &amp;lt; 0.001, respectively). Number of recovered oocytes, embryo number per cycle, cumulative pregnancy rate followed the same trend (p &amp;lt; 0.001). The decrease in CLBR with increasing female age was significantly correlated with fertilization rate and CLBR in all three female age groups (P &amp;lt; 0.001). Finally, to further control for possible patient-specific confounding factors, maternal age, number of retrieved oocytes, percent of inseminated oocytes and fertilization rate were evaluated in a multivariate logistic regression analysis. From this assessment, fertilization rate emerged as a factor independently associated with cumulative live birth rate, to a degree equivalent or higher compared with the number or retrieved oocytes. Limitations, reasons for caution The study design is retrospective and requires further refinement to control for factors that may impact clinical outcome. Wider implications of the findings These data indicate a positive association of FR with CLBR, thereby suggesting that fertilization, in addition to representing an assay for gamete quality and laboratory performance,has an independent clinical significance.Irrespective of the number of retrieved oocytes and female age, we observed that, rates of FR are positively associated with CLBR. Trial registration number None

P–090 Cumulative live birth rates (CLBR) per embryo transfer, embryo transferred and inseminated oocyte are not affected by paternal age in infertile male donor egg cycles

Abstract Study question Is reproductive success measured as CLBR per inseminated oocyte, per embryo transfer and per embryo transferred affected by paternal age in donor egg IVF-ICSI cycles? Summary answer Paternal age does not significantly affect reproductive outcomes measured by CLBR per inseminated oocyte, embryo transfer and embryo transferred in donor egg IVF-ICSI cyles. What is known already In recent years, the delay in the start of formation of a family has led to an increase of the average male age at which the first child is conceived. Therefore, there is a growing interest on the study of the impact of male age on the reproductive outcomes in assisted reproduction cycles (ART). Several studies have evaluated the effect of paternal age on reproductive outcomes using donor egg cycles to control for female factors. However, the results obtained on this topic are still controversial leading to a need of more research about it, which this study tries to address. Study design, size, duration This retrospective observational multicentric cohort study has included donor IVF-ICSI treatments (n = 1539) performed to couples with etiology of male infertility (non-normozoospermic) in Spain IVIRMA clinics between January 2008 and March 2020 using patients’ own sperm sample. Paternal age ranged from 28 to 74 years. The study population was categorized in 5 groups following the criterion of homogenizing the number of observations between groups 28–38 (A), 38–41 (B), 41–44 (C), 44–48 (D) y 48–74 (E) years. Participants/materials, setting, methods Considering that male age could be a factor affecting reproductive outcomes, we evaluated men with different age that performed a donor IVF-ICSI treatment with their own semen, etiology of male infertility and known age. Data was exported in order to obtain the clinical database and Kaplan-Meier was used for data analysis. P &amp;lt; 0.05 was considered statistically significant. We measured reproductive success by CLBR per embryo transfer, per embryo transferred and per inseminated oocytes until live birth. Main results and the role of chance This study considered approximately 836 patients and 1411 embryo transfers. The CLBR per inseminated oocyte showed no significant difference between the study groups: A (3.09%, 42.61%, 71.96%), B (4.53%, 39.76%, 84.19%), C (5.89%, 47.04%, 78.61%), D (2.99%, 46.7%, 73.15%) and E (3.89%, 38.39%, 78.85%) for 7, 12 and 17 inseminated oocytes, respectively. In terms of CLBR per embryo transfer, the results obtained for each of the age groups were: A (51.55%, 70.69%, 92.18%), B (50.40%, 78.13%, 100.00%), C (53.68%, 71.69%, 100.00%), D (51.71%, 79.72%, 100.00%) and E (46.69%, 60.02%, 70.01%) for 3, 5 and 7 embryo transfers, respectively. No statistically significant differences were found among the studied age groups. CLBR per embryo transferred also did not show statistically significant differences between the age groups: A (8.43%, 53.34%, 72.13%), B (8.55%, 44,67%, 71.65%), C (10.40%, 53.94%, 72.49%), D (7.25%, 43.61%, 75.12%) and E (8.21%, 45.99%, 64.55%) for 1, 4 and 7 embryos transferred, respectively. Therefore, no significant differences were found in the number of inseminated oocytes, embryo transfers and embryos transferred needed to achieve a live birth between the age groups (p &amp;gt; 0.05), suggesting that maybe paternal age has no relevant clinical effect on donor egg cycles with our categorization. Limitations, reasons for caution The retrospective nature of this study leads to biases derived from the clinical practice and to the presence of missing data (limiting sample size). Moreover, this study included donor egg cycles for controlling female factors, so this limits the generalization of our results to a population of young women. Wider implications of the findings: Our study showed that the reproductive success measured as CLBR per embryo transfer, embryo transferred and inseminated oocytes was not statistically significant different among the studied age groups in donor egg cycles. Therefore, considering our study setting, paternal age does not affect reproductive success, however further studies should be done. Trial registration number NA

P–087 Paternal age clinical effect on cumulative live birth rates (CLBR) per inseminated oocyte, embryo transfer and embryo transferred in autologous IVF-ICSI cycles of infertile males

Abstract Study question Is reproductive success measured as CLBR per inseminated oocyte, per embryo transfer and per embryo transferred affected by paternal age in autologous IVF-ICSI cycles? Summary answer The number of embryo transfers and embryos transferred until live birth, but not the number of inseminated oocytes, were significantly different among the age groups. What is known already In recent years, there has been an increase of the average paternal age at which the first child is conceived. Therefore, there is a growing interest on the study of the impact of male age on the reproductive outcomes in assisted reproduction cycles (ART). Several studies have shown negative effects of advanced paternal age on semen parameters, embryo aneuploidy, miscarriage, male infertility,.... However, other studies have found no association between them. Hence, the impact of paternal age on reproductive outcomes still remains uncertain, leading to a need of more research on this topic, which this study tries to address. Study design, size, duration This retrospective observational multicentric cohort study has included autologous IVF-ICSI treatments (n = 6295) performed to couples with etiology of male infertility (non-normozoospermic) in Spain IVIRMA clinics between January 2008 and March 2020 using patients’ own sperm sample. Paternal age ranged from 20 to 75 years. The study population was categorized in 5 groups following the criterion of homogenizing the number of observations between groups: 20–34 (A), 34–37 (B), 37–39 (C), 39–42 (D) and 42–75 (E) years. Participants/materials, setting, methods Considering that male age could be a factor affecting reproductive outcomes, we evaluated men with different age that performed an autologous IVF-ICSI treatment with their own semen, etiology of male infertility and known age. Data was exported in order to obtain the clinical database and Kaplan-Meier was used for data analysis. P &amp;lt; 0.05 was considered statistically significant. We measured reproductive success by CLBR per embryo transfer, per embryo transferred and per inseminated oocytes until live birth. Main results and the role of chance This study considered approximately 2976 patients and 4385 embryo transfers. The CLBR per inseminated oocyte showed no significant difference between the study groups: A (6.43%, 48.24%, 81.38%), B (5.74%, 52.14%, 82.87%), C (6.14%, 49.83%, 83.69%), D (5.89%, 53.60%, 81.16%) and E (6.61%, 47, 52%, 77.85%) for 4, 13 and 21/22 inseminated oocytes, respectively. In terms of CLBR per embryo transfer, the results obtained for each of the age groups were: A (31.81%, 71.89%, 87.63%), B (28.89%, 67.87%, 82.63%), C (27.10%, 68.87%, 88.17%), D (23.45%, 64.63%, 100.00%) and E (22.88%, 55.48%, 63.31%) for 1, 4 and 7 embryo transfers, respectively. There were statistically significant differences in the CLBR per embryo transfer between the studied age groups (p &amp;lt; 0.0001). CLBR per embryo transferred for each age group was as follows: A (10.85%, 60.53%, 80.88%), B (9.34%, 59.75%, 78.23%), C (11.89%, 57.63%, 74.97%), D (10.25%, 52.71%, 77.76%) and E (11.71%, 51.50%, 71.51%) for 1, 4 and 7 embryos transferred, respectively. As in the case before, there were statistically significant differences in the CLBR per embryo transferred between the age groups (p &amp;lt; 0.05). The findings presented highlight that the increase in paternal age could be affecting the reproductive outcomes in IVF-ICSI cycles using autologous oocytes. Limitations, reasons for caution The retrospective nature of this study leads to biases derived from the clinical practice and to the presence of missing data (limiting sample size). Moreover, this study considered autologous cycles, therefore the results were not adjusted for female factors. Wider implications of the findings: Our study showed that reproductive outcomes measured by CLBR per embryo transfer and embryo transferred until live birth were significantly different between the paternal age groups in autologous IVF-ICSI cycles of infertile males. Hence, paternal age could be affecting reproductive outcomes and it should be considered for improving infertility counselling. Trial registration number NA

P-382 Pre-selected for an award: Association of extended culture to blastocyst and gestational trophoblastic disease risk following IVF/ICSI assisted reproduction cycles: An analysis of large UK National database

Abstract Study question Is there any association between stage of embryo at transfer based on extended in vitro culture and gestational trophoblastic disease risk during assisted reproduction? Summary answer No significant association between stages of embryo transfer from zygote stage to blastocyst stage was found after analysing 540376 cycles of IVF and ICSI. What is known already Gestational trophoblastic disease (GTD), commonly referred to as molar pregnancy, results from abnormal proliferation of the trophoblast with a reported incidence of ∼1 in 700 in the UK. Despite technological advances such as ICSI, PGT and selection of normally fertilised (2PN) embryos, there are reported cases of GTD following assisted reproduction. Blastocyst transfer is associated with higher pregnancy and live birth rates but evidence is lacking whether extended embryo culture to blastocyst stage influences implantation of an abnormal embryo or abnormal trophoblastic proliferation leading to GTD. Study design, size, duration A retrospective study was carried out using Human Fertilisation and Embryology Authority (HFEA) anonymised register data from 1999 to 2016. HFEA holds the longest running register for fertility treatment data in the world and is the national database for fertility treatment data in UK. A total of 540376 fresh IVF or ICSI assisted reproduction cycles were analysed. Participants/materials, setting, methods There were 1033588 treatment cycles during the study period but only 540376 cycles met the inclusion criteria of fresh IVF or ICSI. Cycles with incomplete data, frozen embryo transfers, donor treatment or surrogacy were excluded. A subgroup analysis of those with primary subfertility was performed after excluding subjects with secondary infertility in order to exclude an effect of a previous molar pregnancy. Multivariate regression analysis was used to adjust for possible confounders. Main results and the role of chance 78 molar pregnancies were reported in the original sample giving a prevalence of 4/10000 live births (78/228461), much lower than the prevalence given with natural pregnancies. Prevalence of molar pregnancy amongst the study population after meeting exclusion criteria was 4 /10000 livebirths (53/156683). Incidence of molar pregnancy was not statistically different between treatment type (0.0001 vs 0.00009). Significantly higher incidence of GTD was seen in the 40 to 42 age category compared to 18-34 category(OR 1.86(95% CI 8.7-3.75)),in par with known higher GTD risk in women above 40 in the general population. Of interesting note, although the incidence of molar pregnancy was significantly lower in women undergoing assisted reproduction increased risk with advancing age is not totally eliminated with treatment. There was no significant association between the occurrence of molar pregnancy with the type and cause for infertility and number of embryos transferred. Crude (1.06 (95% CI 0.852-1.31)) and adjusted odds ratios (1.07 (95% CI (0.857-1.32)) did not show any association between day of embryo transfer and molar pregnancy even after adjusting for age and excluding secondary infertility. Selection of blastocyst stage embryo after extended culture did not alter the likelihood of having a GTD compared to cleavage stage embryo. Limitations, reasons for caution The retrospective analysis of anonymised HFEA data limited adjustments for confounders such as smoking, previous history of GTD, ethnicity etc that predispose to GTD. Caution needs to be exercised for under-reporting of GTD to HFEA and lack of information on type of GTD identified. Wider implications of the findings Though GTD cannot be prevented by IVF/ICSI, the incidence is significantly low and extended culture is not associated with higher risk of abnormal trophoblastic proliferation or GTD occurrence with IVF/ ICSI treatment. These findings would aid informed implications counselling and reassurance of patients during assisted reproduction treatments. Trial registration number not applicable

P–235 Are morphokinetics parameters and profitability of an assisted reproduction cycle related to sperm selection technique? Microfluidic sperm sorter chip versus magnetic activated cell sorting

Abstract Study question Does microfluidic sperm sorter offer any biological improvement over magnetic activated cell sorting (MACS)? Summary answer Microfluidic approach for selecting high profile spermatozoa is as good as magnetic activated cell sorting in terms of morphokinetic, fertilization and good quality blastocyst rate. What is known already Microfluidic sperm sorter chip is a method for select non-fragmented DNA sperm. We know that the use of these non-fragmented DNA sperms can improve the clinical results and the useful blastocyst rate. As several studies have shown previously, embryos morphokinetics parameters are affected by culture medium, ovarian stimulation, oxygen tension, origin of the oocytes, or the age of the patient, this is why we wanted to compare if the cleavage times using time lapse technology, are different depending on the sperm selection method used to select sperm with the non-fragmented DNA. Study design, size, duration Prospective and observational study performed between May 2019 to January 2021 in IVI Madrid and IVI Sevilla. Seminal samples from couples participating in the study were divided into two aliquots; each of them was processed according to one of the study methods. 53 couples were included in the study. Half of the oocyte from each donor were microinjected with sperm selected through MACS (n = 281) and the other half through a microfluidic device (n = 275). Participants/materials, setting, methods These oocytes were microinjected with both types of sperm samples and incubated in EmbryoScope. Cellular events studied in this study included cellular divisions until blastocyst stage, appearance and fading of some cellular structures and the duration of the first, second and third cellular cycle (cc1, cc2 and cc3) as well as their synchrony (S1, S2, S3). Data were exported from the EmbryoViewer data base. We perform an ANOVA statistical analysis to analyze the data. Main results and the role of chance No significant differences between both sperm selection methods were found regarding the time of cell division (from T2 to Tblstocyst), the cellular cycles duration (cc1, cc2 and cc3) or the synchrony of the cellular cycles divisions (s1, s2 and s3). However, a clear trend towards statistical significance has been found in both duration of cc2 (p = 0.052) being longer in MACS embryos than in microfluidic sperm sorting embryos, and in the expansion of the blastocyst, which occurs earlier in embryos that come from MACS than in those that come from microfluidic sperm sorting (p = 0.097). These two events could indicate a better embryo cleavage dynamic in the case of MACS embryos, with a better blastocyst expandability and the necessary time to carry out all the biological events that must occur in the cc2. However, significant difference was found in the direct cleavage from 1 to 3 cells embryo stage, which is one of the adverse events that more affects embryo implantation, being higher in microfluidic sperm sorting group (p = 0,037). Finally, the fertilization rate (73.1% vs 76.9%) and the good quality blastocyst rate (53.7% vs 56.5%) were higher in MACS embryos than in microfluidic sperm sorting embryos, although no significant differences were found. Limitations, reasons for caution This study has been performed in donated oocytes, so these results may not be extrapolated to other groups of assisted reproduction patients. However, more data are needed to draw firm conclusions. Furthermore, it´s crucial to increase the sample size to check if the trends founded reach statistical significance. Wider implications of the findings: Microfluidic sorting of unprocessed semen in un unselected population is as efficacious as magnetic activated cell sorting according embryo morphokinetic, fertilization rate and useful blastocyst rate. Microfluidic sperm sorting does not show clinical advantage over MACS considering this data collection. Trial registration number NCT04061484

Predictive value of serum and follicular fluid chemerin concentrations during assisted reproductive cycles in women with polycystic ovary syndrome

Aims To evaluate the effect of serum and follicular fluid (ff) Chemerin levels on Assisted Reproductive Technology (ART) outcomes in lean patients with PCOS. Materials and methods The study included 76 infertile reproductive aged women, between 21–35 years who underwent intracytoplasmic sperm injection (ICSI) procedure. Serum and ff Chemerin levels were evaluated. Fertilization and clinical pregnancy rate were compared between the groups. Results Serum (13.32 ng/ml versus 29.82 ng/ml) and ff chemerin (35.90 ng/ml versus 87.60 ng/ml) levels were significantly higher in lean PCOS patients compared to controls (p < .01). Serum (24.5 ng/ml versus 18.4 ng/ml) and ff chemerin (71.7 ng/ml versus 52.8 ng/ml) levels were higher in subjects without clinical pregnancy compared to the subjects with clinical pregnancy (p < .05). A cutoff value of 36.2 ng/ml in the ff chemerin level was found to estimate clinical pregnancy with 83% sensitivity and 52% specificity (Area under the curve 0.66; 95% confidence interval, 0.53–0.79). A cutoff value of 12.7 ng/ml in the serum chemerin level was found to estimate clinical pregnancy with 91% sensitivity and 49% specificity (Area under the curve 0.65; 95% confidence interval, 0.52–0.78). Clinical pregnancy rates were significantly higher in group with lower serum chemerin levels (80.0% versus 30.4%, p < .001). High serum chemerin levels are associated with failure of assisted reproduction [OR:0.1(95% CI, 0.03–0.4, p < .001)]. Conclusions PCOS is associated with higher serum and ff chemerin levels and high serum chemerin level is a risk factor for failed ART cycle.

P50: Endometrium in patients with recurrent implantation failures in assisted reproduction cycles – Immune, morphological and genetic pattern

P50: Endometrium in patients with recurrent implantation failures in assisted reproduction cycles – Immune, morphological and genetic pattern

New concepts and difficulties with progesterone supplementation in the luteal phase.

Management of the luteal phase (LP) in assisted reproductive cycles has aroused interest in recent years. The reason is that it seems that the individualization of LP support may be necessary, since the concept of 'one size fits all' does not apply to this treatment. Studies carried out in hormone replacement therapy cycles (also called artificial cycles) have shown that serum levels of progesterone (P) are related to pregnancy outcomes. This represents a milestone in the management of artificial cycles (AC), because until a few years ago it was believed that serum levels did not really reflect the effectiveness of P, which is why they were neglected. However, it is not as straightforward as it seems, because the interpretation of serum progesterone values will depend on the type of progesterone used and its route of administration. Likewise, the findings observed in AC are not applicable to what occurs in a fresh transfer cycle after ovarian stimulation or an embryo transfer in the context of a natural cycle. In this manuscript, we will summarize the current situation in LP management.