Fertilization Rates In IVF Research Articles

P-762 Discovery and quantification of microplastics in human cumulus granulosa cells

Abstract Study question The impact of microplastics (MPs) on human health has received increasing attention; their accumulation in female reproductive system and possible harms have not been quantified. Summary answer This study firstly reveals the contamination of human cumulus granulosa cells and follicular fluid by MPs and the effects of these contaminants on ART outcomes. What is known already MPs are ubiquitous in soil, sea, air, fresh water, and other environments. MPs are ingested by humans through the food chain, air and direct contact, resulting in various health problems, such as endocrine disruption, immune disturbance and reproductive toxicity. MPs exposure has been found to have a great impact on females, as MPs may accumulate in the ovaries and reduce the number of follicles, ovarian size, pregnancy rate, embryo production and fertility. To date, one study has found evidence of MPs in human placentas for the first time, but MPs contamination in other human reproductive systems has not been identified. Study design, size, duration The inclusion criteria comprised female patients of infertile couples excluding male factors who visited the outpatient department of the Reproductive and Genetic Hospital of CITIC-Xiangya between January 2019 and January 2021.We randomly selected 16 women who met the criteria and assessed their CCs and follicular fluid samples for the presence of MPs. Participants/materials, setting, methods In the present study, 16 cumulus granulosa cell samples and 2 follicular fluid samples were collected, and MPs were detected via pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). Main results and the role of chance MPs were detected in both cumulus granulosa cells and follicular fluid, with an average abundance of 313.14±246.69 mg/kg in cumulus granulosa cellsand 12.88±15.54 mg/kg in follicular fluid. Polyethylene terephthalate was the predominant polymer in cumulus granulosa cells. There were no significant differences in the IVF fertilization rates, ICSI fertilization rates, IVF good-quality embryo rates, or ICSI good-quality embryo rates between the high-MP group and low-MP group. However, embryos in the high-MP group exhibited a lower normal cleavage rate than did those in the low-MP group, while the embryos in the high-MP group exhibited a greater proportion of noncleavage than did those in the low-MP group. Limitations, reasons for caution It is not clear how long these particles may persist in the human body. The method we used can detect only the concentration of MPs but not the size of MP particles. Moreover, our method cannot be used to quantify smaller-diameter nanoplastics, which are considered to pose greater risks. Wider implications of the findings This is the first study in which MPs were identified and characterized in human ovarian granulosa cells, providing reference data for evaluating their toxicity to the human female reproductive system. Trial registration number Not applicable

Treatment with cholesterol just after thawing maintains the fertility of bull sperm.

Freezing and thawing diminish sperm motility and fertility by disrupting the cholesterol balance in sperm plasma and organelle membranes. The aim of this study was to elucidate the mechanisms through which exogeneous cholesterol treatment enhances the quality of frozen-thawed bull sperm. The incorporation of cholesterol was investigated using boron-dipyrromethene (BODIPY)-cholesterol, and BODIPY signals were detected not only in the plasma membrane but also in the midpiece region immediately after thawing. The positive signal of cholesterol in the midpiece region was inhibited by a scavenger receptor class B Type I (SR-BI) inhibitor, block lipid transport 1 (BLT-1). To comprehend the role of exogenous cholesterol in the functions of the plasma membrane, propidium iodide (PI)/Annexin V and peanut agglutinin lectin (PNA) staining were performed. The results showed that treatment with exogenous cholesterol increased the number of acrosome-intact sperm and decreased the number of sperm with damage to the plasma membrane. Moreover, since BODIPY signals were also observed in the midpiece region, mitochondrial function was evaluated using a flux analyzer and a flow cytometer with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) staining, revealing an increase in the number of sperm with high-mitochondrial activity and oxygen consumption. Finally, to assess sperm fertility, computer-assisted sperm analysis (CASA) and IVF were carried out. Sperm velocities and fertilization rates in IVF were significantly enhanced by the addition of cholesterol just after thawing. Thus, the treatment with cholesterol after thawing protected the plasma membrane from the stress of thawing and maintained mitochondrial function, thereby preserving the fertilization ability of frozen-thawed bull sperm for conventional IVF and artificial insemination (AI). Therefore, the application of cholesterol just after thawing is a promising option for improving the fertility of frozen-thawed sperm.

P-096 Prediction of fertilization success by voltage-dependent intracellular Ca2+ dynamics in non-normozoospermic human semen samples undergoing in vitro fertilization: preliminary results

Abstract Study question Does the change in intracellular calcium levels evoked by artificial membrane depolarizations predict fertilization success in a non-normozoospermic semen sample undergoing assisted reproductive techniques? Summary answer The increase in the intracellular calcium after artificial plasma membrane depolarization correlates negatively with fertilization rates in conventional IVF but not with ICSI What is known already Sperm capacitation-related features, like the regulation of the intracellular calcium concentration ([Ca2+]i) and the hyperpolarization of the plasma membrane potential (Vm), have been proposed as predictors of success during artificial reproduction techniques (ART). Intriguingly, upon Vm depolarization, sperm exhibit a rapid and transitory increase in the [Ca2+]i controlled, likely, by the activation of the sperm-specific calcium channel CatSper. The regulation of CatSper activity is essential for sperm function and is crucial for fertilization. Therefore we wondered if the Vm depolarization-dependent rise in the [Ca2+]i can be used to predict fertilization success rates in non-normozoospermic semen samples of men undergoing ART. Study design, size, duration This prospective study employed unidentified semen samples from 29 non-normozoospermic men (37.8 ± 5.6 years old) undergoing fertility treatment from March 2022 to the present at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and procedures with less than 3 oocytes were excluded. Ten normozoospermic semen samples were analyzed as a control. The depolarization-dependent [Ca2+]i changes were evaluated by flow cytometry under four different Vm conditions: resting (variable among samples), –64, –46, and –32 mV. Participants/materials, setting, methods Semen samples were obtained on the same day as oocyte retrieval. Sperm cells were washed and incubated in capacitating HTF media for further use in conventional IVF or ICSI procedures. When available, the surplus cells were stained with vitality, Ca2+, and Vm-sensitive fluorescent dyes. Fluorescence changes were evaluated by AccuriC6 flow cytometer. The kinetical analysis of the transitory [Ca2+]i increase as well as the statistical comparisons were performed using custom-made R programming language code. Main results and the role of chance Analysis of resting Vm showed that normozoospermic semen samples presented more depolarized Vm than non-normozoospermic controls (–75.7 ± 6.9 vs –67.3 ± 10.5 mV, respectively, p = 0.02). The former group of semen samples was either used for conventional IVF (n = 11) or ICSI (n = 14). The semen samples in these last categories showed a tendency to present a more depolarized Vm than non-normozoospermic samples (–69.9 ± 8.8 and –66.6 ± 11.3 mV, respectively). No differences were seen in the resting Ca2+ levels. We then induced a Vm hyperpolarization by adding the K+ ionophore, valinomycin followed by increasing concentrations of extracellular K+. Each depolarizing stimulus evoked a fast and transitory increase in the [Ca2+]i. We then calculated seven different kinetic parameters of such responses: basal Ca2+ level, increase and decrease velocity, maximum Ca2+ reached, response durability, and area under the curve (AUC). The basal Ca2+ level reached after depolarizing stimuli correlated negatively (R = –0.64, p = 0.03) with the conventional IVF fertilization ratio (number of zygotes/total number of mature oocytes) when the Vm was clamped either to –64 and –46 mV. A negative and significant correlation (R = –0.76, p = 0.006) was seen for the AUC analysis at –64 mV. No relevant correlations were found in the ICSI samples. Limitations, reasons for caution This is an in vitro study, and caution must be taken when extrapolating these results in vivo. The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples (in progress). Wider implications of the findings Further research into the molecular regulation of the CatSper channel could offer promising alternatives for clinicians to appropriately choose between IVF and ICSI for each couple. This protocol is fast and simple to perform in a laboratory equipped with a flow cytometer or any fluorescence-based reader. Trial registration number NA

P-094 Dynamics of progesterone-induced intracellular Ca2+ changes in sperm predicts zygote formation rate by IVF: Preliminary results

Abstract Study question Can the overall intracellular Ca2+ dynamics evoked by progesterone stimulation predict the fertilizing potential of a non-normozoospermic semen sample under fertility treatment? Summary answer Some kinetic parameters of the transient intracellular Ca2+ increase evoked by progesterone stimulation correlate with fertilization rates in conventional IVF but not with ICSI. What is known already Stimulation with female hormone progesterone induces the activation of the sperm-specific ion channel CatSper which in turn produces a fast increase in the intracellular Ca2+ concentration ([Ca2+]i). After reaching a maximum [Ca2+]i, different Ca2+ clearance mechanisms become active producing a transient followed by a plateau. We investigated whether the [Ca2+]i dynamics upon progesterone stimulus may be used to assess the fertilizing potential of sperm samples from men undergoing assisted reproduction. Study design, size, duration This prospective study employed unidentified semen samples from 39 non-normozoospermic men (age 38 ± 6 years old) undergoing fertility treatment from March 2022 to the present at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and ART procedures with less than 3 oocytes were excluded. Ten normozoospermic semen samples were also analyzed. The progesterone-dependent [Ca2+]i changes were evaluated using time-lapse flow cytometry. Participants/materials, setting, methods Semen samples were obtained on the same day as oocyte retrieval. Sperm cells were washed and incubated in capacitating HTF media for further use in conventional IVF or ICSI procedures. When available, the surplus cells were stained with vitality and Ca2+-sensitive fluorescent dyes. Fluorescence changes were evaluated by AccuriC6 flow cytometer. Progesterone-induced [Ca2+]i transient as well as the correlations with the fertilization rates were calculated using custom-made R programming language code. Main results and the role of chance The progesterone (1 µM) stimulation evoked a fast and transitory increase in the [Ca2+]i. We then calculated six different kinetics parameters of such responses: basal Ca2+ level, increase and decrease in velocity, maximum Ca2+ level, total response duration, and the area under the curve (AUC). There were no significant differences in any of these parameters between normozoospermic donors (and non-normozoospermic patients. The former group of semen samples were divided into either conventional IVF (n = 17) or ICSI (n = 21). In IVF but not ICSI samples, the duration, and the area under the curve of the progesterone response tended to be higher than in normozoospermic (2.39±0.7 min, p = 0.03, and 113±30 AUF*min, p = 0.18, respectively). Furthermore, both parameters showed a tendency to correlate negatively with the conventional IVF (R= –0.45, p = 0.07 and R= –0. 5, p = 0.04, respectively) but not with ICSI fertilization ratio (number of zygotes/total number of mature oocytes). These results suggest that cells with a better Ca2+ recovery mechanism are well suited for IVF. Interestingly, we observed no difference between the magnitude of the [Ca2+]i increase after progesterone stimulus among donors, IVF, or ICSI samples, as was previously reported. Differences in the concentration of progesterone could explain such results. Limitations, reasons for caution The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples (in progress). It is well known that progesterone induces variable responses even among the same donor cells. Our population-based analysis may be underestimating such single cells variations. Wider implications of the findings Current protocols for sperm analysis fail to successfully predict the fertilizing capability of semen samples from men seeking reproductive treatment. Further research into the molecular regulation of Ca2+ dynamics could offer promising alternatives for clinicians to appropriately choose between IVF and ICSI for each couple. Trial registration number .

Elevated and Sustained Intracellular Calcium Signalling Is Necessary for Efficacious Induction of the Human Sperm Acrosome Reaction.

Progesterone and prostaglandin E1 are postulated to trigger the human sperm acrosome reaction (AR). However, their reported efficacy is very variable which likely, in part, reflects the plethora of experimental conditions and methodologies used to detect this physiologically relevant event. The purpose of this study was to develop an assay for the robust induction and objective measurement of the complete AR. Sperm from healthy volunteers or patients undertaking IVF were treated with a variety of ligands (progesterone, prostaglandin E1 or NH4Cl, alone or in combinations). AR, motility and intracellular calcium measurements were measured using flow cytometry, computer-assisted sperm analysis (CASA) and fluorimetry, respectively. The AR was significantly increased by the simultaneous application of progesterone, prostaglandin E1 and NH4Cl, following an elevated and sustained intracellular calcium concentration. However, we observed notable inter- and intra-donor sample heterogeneity of the AR induction. When studying the patient samples, we found no relationship between the IVF fertilization rate and the AR. We conclude that progesterone and prostaglandin E1 alone do not significantly increase the percentage of live acrosome-reacted sperm. This assay has utility for drug discovery and sperm toxicology studies but is not predictive for IVF success.

Effect of early rescue ICSI and split IVF-ICSI in preventing low fertilization rate during the first ART cycle: A real-world retrospective cohort study.

PurposeTo determine the utility of short gamete coincubation in in vitro fertilization (IVF‐S) combined with early rescue intracytoplasmic sperm injection (R‐ICSI) and split IVF‐ICSI in preventing low fertilization based on a retrospective cohort study.MethodsCouples with a high risk of low IVF fertilization during the first ART cycle underwent IVF‐S with R‐ICSI or split IVF‐ICSI. Fertilization rate, embryo quality, and clinical outcomes were measured.ResultsAfter propensity score matching, we included 188 couples in the IVF‐S with R‐ICSI group as Group 1 and 720 in the split IVF‐ICSI group as Group 2. Normal fertilization rates were similar; however, Group 1 had a higher multiple pronuclei rate (10.42% vs. 4.50%, p < 0.001) but a higher embryo utilization rate (59.84% vs. 53.60%, p < 0.001). The groups were similar in the rates of high‐quality embryos, embryo implantation, clinical pregnancy, and live birth. Low IVF fertilization rate was 4.79% and 9.03% in Group 1 and Group 2, respectively, with similar fertilization rate and embryo development.ConclusionIVF‐S with early R‐ICSI and split IVF‐ICSI were effective strategies in preventing low fertilization rate. IVF‐S with early R‐ICSI could become the preferred approach because of its advantages—higher embryo utilization rate, fewer ICSI procedures, similar clinical pregnancy rate, and live birth rate.

Lower Spermatozoal PIWI-LIKE 1 and 2 Transcript Levels Are Significantly Associated with Higher Fertilization Rates in IVF.

The four human PIWI-LIKE gene family members PIWI-LIKE 1–4 play a pivotal role in stem cell maintenance and transposon repression in the human germline. Therefore, dysregulation of these genes negatively influences the genetic stability of the respective germ cell and subsequent development and maturation. Recently, we demonstrated that a lower PIWI-LIKE 2 mRNA expression in ejaculated spermatozoa is more frequent in men with oligozoospermia. In this study, we analysed how PIWI-LIKE 1–4 mRNA expression in ejaculated spermatozoa predicts ART outcome. From 160 IVF or ICSI cycles, portions of swim-up spermatozoa used for fertilization were collected, and the total RNA was isolated. PIWI-LIKE 1–4 mRNA expression was measured by qPCR using TaqMan probes with GAPDH as a reference gene. PIWI-LIKE 1 and 2 transcript levels in the spermatozoa of the swim-up fraction were positively correlated to each other (rS = 0.78; p < 0.001). Moreover, lower PIWI-LIKE 2 mRNA levels, as well as lower PIWI-LIKE 1 mRNA levels, in these spermatozoa were positively associated with a fertilization rate ≥ 50% in the respective ART cycles (p = 0.02 and p = 0.0499, Mann–Whitney U-Test). When separately analysing IVF and ICSI cycles, PIWI-LIKE 1 and 2 transcript levels were only significantly associated to increased fertilization rates in IVF, yet not in ICSI cycles. Spermatozoal PIWI-LIKE 3 and 4 transcript levels were not significantly associated to fertilization rates in ART cycles. In conclusion, lower levels of spermatozoal PIWI-LIKE 1 and 2 mRNA levels are positively associated with a higher fertilization rate in IVF cycles.

P–064 Application of an artificial intelligence model for morphologic prediction of fertilization-competent human spermatozoa

Abstract Study question What is the role of artificial intelligence in selecting fertilization-competent human spermatozoa according to their morphological characteristics? Summary answer: The established AI model in this study can be potentially used to select semen samples with superior fertilization potential in clinical settings. What is known already Defective spermatozoa-zona pellucida (ZP) interaction causes subfertility and is a major cause of low IVF fertilization rates. While ICSI benefits patients with defective spermatozoa-ZP binding, a standard method to identify such patients prior to conventional IVF is lacking. The application of artificial intelligence to sperm morphology analysis has become a topic of growing interest owing to the fact that the conventional assessment is highly subjective and time-consuming. Deep-learning, a core element of artificial intelligence (AI), incorporates the convolutional neural networks (CNN) to process all the data composing a digital image through successive layers to identify the underlying pattern. Study design, size, duration The fertilization-competent spermatozoa were isolated according to their binding ability to the ZP. The ZP-bound and -unbound spermatozoa were collected for functional assays and to establish an AI model for morphologic prediction of sperm fertilization potential. Human spermatozoa (n = 289) were isolated from normozoospermic samples. Human oocytes (n = 562) were collected from an assisted reproduction program in Hong Kong. Sample collection has been ongoing and will continue until the end of this study in November 2021. Participants/materials, setting, methods Sperm-ZP binding assay was employed to collect ZP-bound and -unbound spermatozoa. The fertilization potential and genetic quality of the collected spermatozoa were evaluated by our established protocols. Diff-Quik- stained images of ZP-bound and -unbound spermatozoa were collected respectively for the establishment of an AI model. A novel algorithm for sperm image transformation and segmentation was developed to pre-process the images. CNN architecture was then applied on these pre-processed images for feature extraction and model training. Main results and the role of chance Our result showed that the sperm-ZP binding assay had no detrimental effect on sperm viability when compared with the raw samples and unbound-sperm subpopulations. ZP-bound spermatozoa were found with statistically higher acrosome reaction rates, improved DNA integrity, better morphology, lower protamine deficiency and higher methylation level when compared with the unbound spermatozoa. A deep-learning model was trained and validated by analyzing a total of 1,334 and 885 of ZP-bound/unbound spermatozoa to evaluate the predictive power of sperm morphology for ZP binding ability. Our newly trained AI-based model showed initial success in classifying the ZP-bound/ unbound spermatozoa according to their morphological characteristics with high accuracy of 85% and low computational complexity. Limitations, reasons for caution This sperm selection method requires micromanipulation and relatively long processing time to recover ZP-bound spermatozoa. In addition to limited availability, the use of human materials may result in interassay variations affecting the reproducibility of this method among laboratories. Wider implications of the findings: In light of current findings, AI-based sperm selection method may provide high predictive values of sperm fertilization potential for clinical purposes. This method is particularly applicable to patients who had poor fertilization outcomes after conventional IVF treatments or those with high degree of defective sperm-ZP binding ability. Trial registration number Not applicable

P-064 Application of an artificial intelligence model for morphologic prediction of fertilization-competent human spermatozoa

Abstract Study question What is the role of artificial intelligence in selecting fertilization-competent human spermatozoa according to their morphological characteristics? Summary answer The established AI model in this study can be potentially used to select semen samples with superior fertilization potential in clinical settings. What is known already Defective spermatozoa-zona pellucida (ZP) interaction causes subfertility and is a major cause of low IVF fertilization rates. While ICSI benefits patients with defective spermatozoa-ZP binding, a standard method to identify such patients prior to conventional IVF is lacking. The application of artificial intelligence to sperm morphology analysis has become a topic of growing interest owing to the fact that the conventional assessment is highly subjective and time-consuming. Deep-learning, a core element of artificial intelligence (AI), incorporates the convolutional neural networks (CNN) to process all the data composing a digital image through successive layers to identify the underlying pattern. Study design, size, duration The fertilization-competent spermatozoa were isolated according to their binding ability to the ZP. The ZP-bound and -unbound spermatozoa were collected for functional assays and to establish an AI model for morphologic prediction of sperm fertilization potential. Human spermatozoa (n = 289) were isolated from normozoospermic samples. Human oocytes (n = 562) were collected from an assisted reproduction program in Hong Kong. Sample collection has been ongoing and will continue until the end of this study in November 2021. Participants/materials, setting, methods Sperm-ZP binding assay was employed to collect ZP-bound and -unbound spermatozoa. The fertilization potential and genetic quality of the collected spermatozoa were evaluated by our established protocols. Diff-Quik- stained images of ZP-bound and -unbound spermatozoa were collected respectively for the establishment of an AI model. A novel algorithm for sperm image transformation and segmentation was developed to pre-process the images. CNN architecture was then applied on these pre-processed images for feature extraction and model training. Main results and the role of chance Our result showed that the sperm-ZP binding assay had no detrimental effect on sperm viability when compared with the raw samples and unbound-sperm subpopulations. ZP-bound spermatozoa were found with statistically higher acrosome reaction rates, improved DNA integrity, better morphology, lower protamine deficiency and higher methylation level when compared with the unbound spermatozoa. A deep-learning model was trained and validated by analyzing a total of 1,334 and 885 of ZP-bound/unbound spermatozoa to evaluate the predictive power of sperm morphology for ZP binding ability. Our newly trained AI-based model showed initial success in classifying the ZP-bound/ unbound spermatozoa according to their morphological characteristics with high accuracy of 85% and low computational complexity. Limitations, reasons for caution This sperm selection method requires micromanipulation and relatively long processing time to recover ZP-bound spermatozoa. In addition to limited availability, the use of human materials may result in interassay variations affecting the reproducibility of this method among laboratories. Wider implications of the findings In light of current findings, AI-based sperm selection method may provide high predictive values of sperm fertilization potential for clinical purposes. This method is particularly applicable to patients who had poor fertilization outcomes after conventional IVF treatments or those with high degree of defective sperm-ZP binding ability. Trial registration number not applicable

Analysis of IVF/ICSI Outcomes in Endometriosis Patients With Recurrent Implantation Failure: Influence on Cumulative Live Birth Rate.

ObjectiveTo study the influence of endometriosis activity on the pregnancy outcomes of patients with recurrent implantation failure (RIF) in in-vitro fertilization/intra-cytoplasmic sperm injection (IVF/ICSI) cycles. The pregnancy outcomes were compared between RIF patients with endometriosis who received treatment at different occasions to explore the appropriate treatment plan for these patients and to optimize the pregnancy-support strategies.DesignAmbispective cohort study.MethodsA total of 330 patients with endometriosis were enrolled from 2008 to 2018 and included 1043 IVF/ICSI cycles. All patients were diagnosed with RIF after IVF/ICSI. Patients were assigned to three subtypes according to different control states of endometriosis, including the untreated, early-treatment, and late-treatment groups. The clinical pregnancy rate, live birth rate, and cumulative live birth rate of endometriosis patients with RIF were the main outcomes; additionally, the fertilization rate, available embryonic rate, and high-quality embryonic rate were also compared.ResultsThe early-treatment and late-treatment groups showed higher cumulative live birth rate than the untreated group (early-treated 43.6% vs. late-treated 46.3% vs. untreated 27.7%, P<0.001), though patients in the two treatment groups had higher rates of adenomyosis and ovarian surgery. The two treatment group showed a better laboratory result than the untreated and especially, the early-treatment group. The untreated group (46.24%) had a lower IVF fertilization rate than the treated group (early-treated [64.40%] and late-treated [60.27%] (P<0.001). In addition, the rates of available embryos and high-quality embryos in the early-treated group were much higher those that in the untreated group (90.30% vs. 85.20%, 76.50% vs. 64.47%). Kaplan–Meier curve showed that patients in the untreated group needed a mean of 23.126 months to achieve one live birth; whereas those in the treated group needed a comparatively shorter duration (early-treated: 18.479 ± 0.882 months and late-treated: 14.183 ± 1.102 months, respectively).ConclusionEndometriosis has a negative influence on IVF/ICSI outcome. The control of endometriosis activity can result in a higher cumulative live birth rate in patients. It is necessary for endometriosis patients to receive medical treatment to achieve a better prognosis especially for those with RIF.

Metabolomics shows no impairment of the microenvironment of the cumulus-oocyte complex in women with isolated endometriosis.

Is there any metabolomic evidence of impairment of the cumulus-oocyte complex (COC) microenvironment in the follicular fluid of women with endometriosis? A prospective observational study from January to July 2018 at the Angers University Hospital, France. Seventy-nine women undergoing IVF with or without intracytoplasmic sperm injection (ICSI) were included: 39 for endometriosis-related infertility and 40 controls with other causes of infertility. A targeted quantitative metabolomic and lipidomic analysis was performed. Patient characteristics (age, body mass index, smoking status, hormonal profile and ovarian reserve markers) were comparable between the endometriosis and the control groups. There was no significant difference in the cumulative FSH dose used for stimulation between the endometriosis and the control groups (2732 versus 2257IU, respectively). There were no differences in the oocyte maturity rates (72.2% versus 77.7%), or in the fertilization rates in IVF and ICSI (49.4% versus 50.2% and 76.4% versus 68.8%, respectively) between the endometriosis and control groups. Among the 188 metabolites analysed, 150 were accurately measured. Univariate analysis did not reveal any significant modification of metabolite concentrations, and none of the multivariate models discriminated between the two groups of patients, even when the study was restricted to the most severe form of endometriosis. No specific metabolomic signature of endometriosis was found in the follicular fluid of women undergoing IVF. These results suggest that there is no microenvironmental impairment of the COC in cases of isolated endometriosis among women with infertility.

Serum and follicular fluid fetuin-B levels are correlated with fertilization rates in conventional IVF cycles.

To investigate the relationship between serum/follicular fluid fetuin-B levels and fertilization outcomes in conventional IVF cycles. A prospective cohort study of conventional IVF treatments including 78 cycles with low fertilization rates (two pronuclei [2PN] rate < 30%; LF group) and 104 cycles performed during the same period with 2PN rate > 70% (high fertilization group, HF). To calculate the required sample size, a two-sample t test power analysis was applied to data from our pilot study, using PASS 11.0 software. Fetuin-B was measured using a commercial sandwich enzyme-linked immunosorbent assay. Serum fetuin-B and follicular fluid fetuin-B were positively correlated (r = 0.703, P < 0.001). Compared to the HF group, the LF group had significantly lower levels of fetuin-B, both in serum (5.81 ± 1.53 vs. 7.19 ± 1.42, P < 0.001) and follicular fluid (5.06 ± 1.29 vs. 6.16 ± 1.52, P < 0.001). The serum fetuin-B level from cycles with polypronuclear (PPN) zygotes was significantly lower when compared to cycles without PPN zygotes (6.82 ± 1.65 vs. 6.10 ± 1.43, P = 0.006). However, serum fetuin-B level was not correlated with preimplantation embryo development or clinical pregnancy. Serum fetuin-B level is correlated with fertilization rate in conventional IVF and it may be used as a predictive marker of fertilization in IVF treatment.

Semen parameters on the day of oocyte retrieval predict low fertilization during conventional insemination IVF cycles.

Poor fertilization during conventional IVF is difficult to predict in the absence of abnormal semen parameters; large-scale studies are lacking. The purpose of this study is to evaluate factors associated with low fertilization rates in conventional insemination IVF cycles. A retrospective cohort study evaluating demographic, reproductive evaluation, and IVF cycle characteristics to identify predictors of low fertilization (defined as 2PN/MII ≤ 30% per cycle). Participants were included if they were undergoing their first IVF cycle utilizing fresh autologous oocytes and conventional insemination with male partner's sperm (with normal pretreatment semen analysis). They were randomly divided into a training set and a validation set; validation modeling with logistic regression and binary distribution was utilized to identify covariates associated with low fertilization. Postprocessing sperm concentration of less than 40 million/ml and postprocessing sperm motility < 50% on the day of retrieval were the strongest predictors of low fertilization in the training dataset. Next, in the validation set, cycles with either low postprocessing concentration (≤ 40 million/ml) or low postprocessing progressive motility (≤ 50%) were 2.9-times (95% CI 1.4, 6.2) more likely to have low fertilization than cycles without either risk factor. Furthermore, cycles with low postprocessing concentration and progressive motility were 13.4 times (95% CI 4.01, 45.06) more likely to have low fertilization than cycles without either risk factor. Postprocessing concentration and progressive motility on the day of oocyte retrieval are predictive of low fertilization in conventional IVF cycles with normal pretreatment diagnostic semen analysis parameters.

Day 5 useable blastocyst rate is an early indicator of clinically significant shifts in the embryo culture system

The ability to track performance of an IVF laboratory using statistical process controls has not been validated and remains an important goal for an early warning signal of shifts in laboratory conditions. The Vienna consensus report recently defined Key Performance Indicators (KPIs) for monitoring fresh IVF and ICSI cycles, but the effectiveness of these KPIs for detecting clinically relevant shifts following changes in laboratory processes is unknown1. The aim of this study was to assess whether embryology KPIs detect shifts in laboratory performance that result in changes in clinical outcomes. Retrospective, multicentre, analysis of KPIs for fresh IVF and ICSI cycles. Three consecutive 5-month periods of embryo culture media were investigated 1) embryo culture in Continuous Single Culture Complete (CSCM-C) (Irvine Scientific) 2) embryo culture in G-TL (Vitrolife) 3) embryo culture returned to CSCM-C (Irvine Scientific). Six primary embryology KPIs were tracked for 1523 cycles from June 2016 to August 2017; ICSI fertilization rate, IVF fertilization rate, useable day 5 blastocyst rate, overall useable blastocyst rate, clinical pregnancy rate and cumulative clinical pregnancy rate. Meaningful KPI shifts were identified by upper and lower warning (2-sigma) and control (3-sigma) limits. KPI shifts were analyzed by ANOVA and multivariate regression. During the 15 months analyzed, the novel KPI, useable day 5 blastocyst rate, fell below the 3-sigma lower control limit for 5 months. The decrease in useable day 5 blastocyst rate, from 43% to 32%, occurred following a change from CSCM-C (Irvine Scientific) to G-TL (Vitrolife) and was detected one week after the change (p < 0.0001). Useable day 5 blastocyst rate subsequently recovered after a change back to CSCM-C (p < 0.0001). A decrease in clinical pregnancy rate (54% to 41%) was also observed, but this was not detected until 3 months after the useable day 5 blastocyst rate shift (p = 0.01). Although culture media type independently effected useable day 5 blastocyst rate and clinical pregnancy rate, the cumulative clinical pregnancy rate was similar among the three time periods. Importantly, overall useable blastocyst rate (day 5-7) remained within control limits (52%) for all three periods, indicating that the overall rate alone may not sufficiently monitor embryology laboratory performance. This study validates a statistical KPI monitoring system to provide systematic, early detection of culture condition shifts in ART laboratories. Useable day 5 blastocyst rate is identified as an important KPI, which complements overall useable blastocyst rate (day 5-7).

Single-cell analysis of [Ca2+]i signalling in sub-fertile men: characteristics and relation to fertilization outcome.

STUDY QUESTIONWhat are the characteristics of progesterone-induced (CatSper-mediated) single cell [Ca2+]i signals in spermatozoa from sub-fertile men and how do they relate to fertilizing ability?SUMMARY ANSWERSingle cell analysis of progesterone-induced (CatSper-mediated) [Ca2+]i showed that reduced progesterone-sensitivity is a common feature of sperm from sub-fertile patients and is correlated with fertilization rate.WHAT IS KNOWN ALREADYStimulation with progesterone is a widely used method for assessing [Ca2+]i mobilization by activation of CatSper in human spermatozoa. Although data are limited, sperm population studies have indicated an association of poor [Ca2+]i response to progesterone with reduced fertilization ability.STUDY DESIGN, SIZE, DURATIONThis was a cohort study using semen samples from 21 donors and 101 patients attending the assisted conception unit at Ninewells Hospital Dundee who were undergoing ART treatment. Patients were recruited from January 2016 to June 2017.PARTICIPANTS/MATERIALS, SETTING, METHODSSemen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service (EoSRES) REC1. [Ca2+]i responses were examined by single cell imaging and motility parameters assessed by computer-assisted sperm analysis (CASA).MAIN RESULTS AND THE ROLE OF CHANCEFor analysis, patient samples were divided into three groups IVF(+ve) (successful fertilization; 62 samples), IVF-FF (failed fertilization; eight samples) and ICSI (21 samples). A further 10 IVF samples showed large, spontaneous [Ca2+]i oscillations and responses to progesterone could not be analysed. All patient samples loaded with the [Ca2+]i-indicator fluo4 responded to progesterone stimulation with a biphasic increase in fluorescence (transient followed by plateau) which resembled that seen in progesterone-stimulated donor samples. The mean normalized response (progesterone-induced increase in fluorescence normalized to resting level) was significantly smaller in IVF-FF and ICSI patient groups than in donors. All samples were further analysed by plotting, for each cell, the relationship between resting fluorescence intensity and the progesterone-induced fluorescence increment. In donor samples these plots overlaid closely and had a gradient of ≈ 2 and plots for most IVF(+ve) samples closely resembled the donor distribution. However, in a subset (≈ 10%) of IVF(+ve) samples, 3/8 IVF-FF samples and one-third of ICSI samples the gradient of the plot was significantly lower, indicating that the response to progesterone of the cells in these samples was abnormally small. Examination of the relationship between gradient (regression coefficient of the plot) in IVF samples and fertilization rate showed a positive correlation. In IVF-FF and ICSI groups, the proportion of cells in which a response to progesterone could be detected was significantly lower than in donors and IVF (+ve) patients. Approximately 20% of cells in donor, IVF(+ve) and ICSI samples generated [Ca2+]i oscillations when challenged with progesterone but in IVF-FF samples only ≈ 10% of cells generated oscillations and there was a significantly greater proportion of samples where no oscillations were observed. Levels of hyperactivated motility were lower in IVF(+ve) and IVF-FF groups compared to controls, IVF-FF also having lower levels than IVF(+ve).LIMITATIONS, REASONS FOR CAUTIONThis is an in vitro study and caution must be taken when extrapolating these results in vivo.WIDER IMPLICATIONS OF THE FINDINGSThis study reveals important details of impaired [Ca2+]i signalling in sperm from sub-fertile men that cannot be detected in population studies.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by a MRC project grant (MR/M012492/1; MR/K013343/1). Additional funding was provided by Chief Scientist Office/NHS research Scotland.

A study on the relationship between the fertilization rate in IVF and the mechanical energy of sperm

A study on the relationship between the fertilization rate in IVF and the mechanical energy of sperm

Correlation between Follicular Fluid’s Androgen Level and Fertilization Rate in Poor Responder Patients Undergone IVF: A Prospective Cohort Done in Yasmin IVF Clinic, Jakarta, Indonesia

&lt;p&gt;&lt;strong&gt;Introduction &lt;/strong&gt;Androstenedione (A4), testosterone (T), and dehidroepiandrosterone (DHEA) are known to be involved in folliculogenesis and follicular maturity. Lower follicular androgen levels in poor responder due to malfunctioned granulosa and theca cells and decreased inhibin B production will decrease FSH and LH. However, androgenic follicular fluid might also induce follicular atresia, decreased oocytes viability, thus affecting fertilization. The aim of current study is to find the correlation between intra-follicular androgen levels and fertilization rate, specifically in poor responder whom contributed in 84% cancelled cycle.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Material and Methods &lt;/strong&gt;This prospective cohort study was done at Yasmin IVF Clinic, Jakarta, Indonesia, in January 2014-March 2015. Infertile women undergone IVF were asked to participate, grouped into poor responder and other, and the androgen levels in the follicular fluid of each consenting patients were measured.&lt;/p&gt;&lt;p class="DecimalAligned"&gt;&lt;strong&gt;Results &lt;/strong&gt;From total 62 patients, aged 23-47 years old (37.6±5,068), there were 21 poor responders, whereas the other 41 patients with other indication. In poor responder group, levels of androstenedione, testosterone, and DHEA were 50.8 – 272.3 (103.5 ± 59.9), 383.2 – 1747.9 (1114.4 ± 373.2), 11.3 – 454.8 (151.3 ± 96.8), whereas in other group the androgen levels were 44.3 – 326.8 (95.1 ± 61.2), 414.1 – 1463.7 (976.9 ± 258.4), 44.6 – 265.8 (132.7 ± 61.3) with the correlation with fertilization rate 0.609, 0.095, and 0.361 respectively.&lt;/p&gt;&lt;p class="DecimalAligned"&gt; &lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion &lt;/strong&gt;Correlation between follicular androgen levels and fertilization rate found to be not significantly different. The low number of subjects might cause this result, as well as the presence of bias, e.g.male factor and endometriosis might also affect fertilization. A multi-center study with larger sample size added with thorough analysis is needed to reconfirm current data.&lt;/p&gt;

Single nucleotide polymorphisms in the Anti-Müllerian hormone (AMH Ile(49)Ser) and Anti-Müllerian hormone type II receptor (AMHRII -482 A>G) as genetic markers in assisted reproduction technology.

The aim of the study was to evaluate whether the presence Antimullerian hormone (AMH) and Antimullerian hormone type II receptor (AMHRII) single nucleotide polymorphisms (SNPs) Ile(49)Ser and -482A>G respectively are related to the assisted reproduction outcome. A prospective cross-sectional observational study was conducted in order to assess the distribution of AMH and AMHRII SNPs in two cohorts, one of healthy women (N = 100) and the control group and the IVF/ICSI group (N = 151) consisted of women undergoing IVF/ICSI treatment for infertility. Furthermore, a prospective longitudinal observational study was performed on the latter group to assess possible associations of these SNPs with patients' characteristics and controlled ovarian stimulation (COS) and pregnancy outcome. Among non-carriers of the AMH (Ile(49)Ser) polymorphism, basal FSH levels were lower in those with more than two of previous IVF attempts and fertilization rate wasstatistically higher in those with peak serum E2 levels below 1500pg/ml, whereas among non-carriers of the AMHRII (-482 A>G) polymorphism, number of follicles was higher in those with more than two previous IVF attempts and total dose of gonadotropins was lower in those with peak serum E2 levels above 1500pg/ml. There was evidence that in specific subgroups of women undergoing IVF/ICSI, AMH and AMHRII SNPs may be related to patients' characteristics and controlled ovarian stimulation and pregnancy outcome and thus may provide a means for the prediction of ovarian response in specific subgroups of women entering an IVF/ICSI program.

Sperm DNA integrity testing: big halo is a good predictor of embryo quality and pregnancy after conventional IVF.

Sperm DNA integrity is a sperm functional parameter of male fertility evaluation. Two parameters of sperm DNA integrity were observed: DNA damage expressed as DNA fragmentation index (DFI) and percentage of the DNA undamaged spermatozoa expressed as big halo. Halosperm test was used for sperm DNA integrity determination. The aim of this study was to evaluate which DNA integrity parameter is better as an embryo quality and pregnancy prognostic parameter after the conventional IVF. We evaluated two embryo groups (positive and negative group) according to the 3rd day cumulative embryo score. Big halo and DFI, as we expected, showed good correlation (r = -0.69; p < 0.001). Receiver operating characteristic (ROC) analyses show that DFI and big halo are significant (p < 0.001) as prognostic parameters of embryo quality. ROC curves comparison of DFI and big halo revealed the AUC value for big halo to be significantly higher (DFI AUC = 0.71 vs. big halo AUC = 0.83; p = 0.025) than for DFI. Big halo was found to be the only independent predictor of embryo quality. Sperm DNA integrity both parameters are good prognostic parameters of embryo quality after the conventional IVF where big halo seems to be better. ROC analyses show DFI and big halo as significant prognostic parameters for achieved pregnancy (AUC ± SE for DFI was 0.67 ± 0.06 and 0.75 ± 0.06 for big halo). To our knowledge, this is the first study demonstrating the correlation between sperm DNA undamaged rate expressed as big halo parameter and semen characteristics as well as the influence on fertilization rate, embryo quality and pregnancy in conventional IVF.

The clinical significance of calcium-signalling pathways mediating human sperm hyperactivation

STUDY QUESTIONWhat is the prevalence of defects in the Ca2+-signalling pathways mediating hyperactivation (calcium influx and store mobilization) among donors and sub-fertile patients and are they functionally significant, i.e. related to fertilization success at IVF?SUMMARY ANSWERThis study identifies, for the first time, the prevalence of Ca2+ store defects in sperm from research donors, IVF and ICSI patients. It highlights the biological role and importance of Ca2+ signalling (Ca2+ store mobilization) for fertilization at IVF.WHAT IS KNOWN ALREADYSperm motility and hyperactivation (HA) are important for fertility, mice with sperm incapable of HA are sterile. Recently, there has been significant progress in our knowledge of the factors controlling these events, in particular the generation and regulation of calcium signals. Both pH-regulated membrane Ca2+ channels (CatSper) and Ca2+ stores (potentially activating store-operated Ca2+ channels) have been implicated in controlling HA.STUDY DESIGN, SIZE, AND DURATIONThis was a prospective study examining a panel of 68 donors and 181 sub-fertile patients attending the Assisted Conception Unit, Ninewells Hospital Dundee for IVF and ICSI. Twenty-five of the donors gave a second sample (∼4 weeks later) to confirm consistency/reliability of the recorded responses. Ca2+ signalling was manipulated using three agonists, NH4Cl (activates CatSper via pH), progesterone (direct activation of CatSper channels, potentially enhancing mobilization of stored Ca2+ by CICR) and 4-aminopyridine (4-AP) (effect on pH equivalent to NH4Cl and mobilizes stored Ca2+). The broad-spectrum phosphodiesterase inhibitor 3-isobutyl-1-methyxanthine (IBMX), a potent activator of HA was also used for comparison. For patient samples, an aliquot surplus to requirements for IVF/ICSI treatment was examined, allowing direct comparison of Ca2+ signalling and motility data with functional competence of the sperm.MATERIALS, SETTING, METHODSThe donors and sub-fertile patients were screened for HA (using CASA) and changes in intracellular Ca2+ were assessed by loading with Fura-2 and measuring fluorescence using a plate reader (FluoStar).MAIN RESULTS AND THE ROLE OF CHANCEThe relative efficacy of the stimuli in inducing HA was 4-AP >> IBMX > progesterone. NH4Cl increased [Ca2+]i similarly to 4-AP and progesterone but did not induce a significant increase in HA. Failure of samples to generate HA (no significant increase in response to stimulation with 4-AP) was seen in just 2% of research donors but occurred in 10% of IVF patients (P = 0.025). All donor samples generated a significant [Ca2+]i increase when stimulated with 4-AP but 3.3% of IVF and 28.6% of ICSI patients failed to respond. Amplitudes of HA and [Ca2+]i responses to 4-AP were correlated with fertilization rate at IVF (P= 0.029; P = 0.031, respectively). Progesterone reliably induced [Ca2+]i responses (97% of donors, 100% of IVF patients) but was significantly less effective than 4-AP in inducing HA. Twenty seven per cent of ICSI patients failed to generate a [Ca2+]i response to progesterone (P= 0.035). Progesterone-induced [Ca2+]i responses were correlated with fertilization rate at IVF (P= 0.037) but induction of HA was not. In donor samples examined on more than one occasion consistent responses for 4-AP-induced [Ca2+]i (R2 = 0.97) and HA (R2 = 0.579) were obtained. In summary, the data indicate that defects in Ca2+ signalling leading to poor HA do occur and that ability to undergo Ca2+ -induced HA affects IVF fertilizing capacity. The data also confirm that release of stored Ca2+ is the crucial component of Ca2+ signals leading to HA and that Ca2+ store defects may therefore underlie HA failure.LIMITATIONS, REASONS FOR CAUTIONThis is an in vitro study of sperm function. While the repeatability of the [Ca2+]i and HA responses in samples from the same donor were confirmed, data for patients were from 1 assessment and thus the robustness of the failed responses in patients’ needs to be established. The focus of this study was on using 4AP, which mobilizes stored Ca2+ and is a potent inducer of HA. The n values for other agonists, especially calcium assessments, are smaller.WIDER IMPLICATIONS OF THE FINDINGSPrevious studies have shown a significant relationship between basal levels of HA, calcium responses to progesterone and IVF fertilization rates. Here, we have systematically investigated the ability/failure of human sperm to generate Ca2+ signals and HA in response to targeted pharmacological challenge and, related defects in these responses to IVF success. [Ca2+]i signalling is fundamental for sperm motility and data from this study will lead to assessment of the nature of these defects using techniques such as single-cell imaging and patch clamping.STUDY FUNDING/COMPETING INTEREST(S)Resources from a Wellcome Trust Project Grant (#086470, Publicover and Barratt PI) primarily funded the study. The authors have no competing interests.