High prevalence of low prognosis by the POSEIDON criteria in women undergoing planned oocyte cryopreservation.

OBJECTIVE: Planned oocyte cryopreservation (OC) is being increasingly utilized worldwide. However, some women cannot accumulate sufficient oocytes because of poor response to stimulation. The POSEIDON classification is a novel system to classify patients with 'expected' or 'unexpected' inappropriate ovarian response to exogenous gonadotropins. Our study aimed to examine the prevalence of POSEIDON patients among women undergoing planned OC. STUDY DESIGN: We retrospectively reviewed the first cycles of 160 consecutive patients undergoing planned OC. Patients were classified into the four POSEIDON groups or as 'non-POSEIDON' based on age, AMH level and the number of oocytes retrieved. The primary outcome measure was the prevalence of POSEIDON patients. RESULTS: Overall, 63 patients (39.4 %) were classified as POSEIDON patients, 12 in group 1, 12 in group 2, 8 in group 3, and 31 in group 4. Compared to non-POSEIDON patients, POSEIDON patients had increased basal FSH levels and reduced serum AMH levels and antral follicle counts, required higher FSH starting doses and increased gonadotropin requirements and reached lower peak serum estradiol levels. Additionally, POSEIDON patients had a lower number of oocytes retrieved (7.6 ± 3.1 vs.20.2 ± 9.9, p < 0.001) and vitrified (5.8 ± 2.9 vs.14.7 ± 6.8, p < 0.001) than non-POSEIDON counterparts, respectively. CONCLUSION: We found a high prevalence of patients being classified as low prognosis according to the POSEIDON criteria among patients seeking planned OC. POSEIDON patients had increased gonadotropin requirements and a significantly lower number of oocytes retrieved and vitrified. This novel, unexpected finding adds clinically relevant information for counselling and management of patients undergoing planned OC.

Stain-Free Sperm Analysis and Selection for Intracytoplasmic Sperm Injection Complying with WHO Strict Normal Criteria.

This multi-center study evaluated a novel microscope system capable of quantitative phase microscopy (QPM) for label-free sperm-cell selection for intracytoplasmic sperm injection (ICSI). Seventy-three patients were enrolled in four in vitro fertilization (IVF) units, where senior embryologists were asked to select 11 apparently normal and 11 overtly abnormal sperm cells, in accordance with current clinical practice, using a micromanipulator and 60× bright field microscopy. Following sperm selection and imaging via QPM, the individual sperm cell was chemically stained per World Health Organization (WHO) 2021 protocols and imaged via bright field microscopy for subsequent manual measurements by embryologists who were blinded to the QPM measurements. A comparison of the two modalities resulted in mean differences of 0.18 µm (CI -0.442-0.808 µm, 95%, STD-0.32 µm) for head length, -0.26 µm (CI -0.86-0.33 µm, 95%, STD-0.29 µm) for head width, 0.17 (CI -0.12-0.478, 95%, STD-0.15) for length-width ratio and 5.7 for acrosome-head area ratio (CI -12.81-24.33, 95%, STD-9.6). The repeatability of the measurements was significantly higher in the QPM modality. Surprisingly, only 19% of the subjectively pre-selected normal cells were found to be normal according to the WHO2021 criteria. The measurements of cells imaged stain-free through QPM were found to be in good agreement with the measurements performed on the reference method of stained cells imaged through bright field microscopy. QPM is non-toxic and non-invasive and can improve the clinical effectiveness of ICSI by choosing sperm cells that meet the strict criteria of the WHO2021.

The Effect of Syringe-Filling Technique on the Risk for Endophthalmitis after Intravitreal Injection of Anti-VEGF Agents.

PURPOSE: This study aimed to compare the risk for post-injection endophthalmitis between different anti-vascular endothelial growth factor (VEGF) agents and syringe preparation techniques. METHODS: A retrospective study of anti-VEGF injections performed in 3 large ophthalmology departments between 2013 and 2019 was conducted. Injections were categorized according to the drug and the syringe-filling technique - prefilling by a hospital pharmacy, prefilling by a good manufacturing practice (GMP) pharmacy, self-drawing from the vial by the injecting physician, and use of a prefilled syringe. Cases of endophthalmitis were identified, and their rates were analyzed. RESULTS: A total of 197,402 injections were included, and 53 cases of endophthalmitis were identified (0.027% risk). The risk of endophthalmitis following injections with syringes that were prefilled by GMP pharmacies or the manufacturers was significantly lower than that following injections which were self-drawn by the physician (0.019% vs. 0.055%, p < 0.0001). For ranibizumab, risk of endophthalmitis decreased since it became available in a prefilled syringe (0.054% vs. 0.014%, p = 0.066), bordering on statistical significance. CONCLUSIONS: The syringe-filling technique is an important factor determining risk of post-injection endophthalmitis. Use of GMP-grade prefilling by professional pharmacies or the manufacturers significantly reduces this risk and should be the technique of choice for all drugs administered by intravitreal injection.

A randomized controlled trial of vaginal progesterone for luteal phase support in modified natural cycle - frozen embryo transfer.

OBJECTIVE: Our aim was to study whether luteal phase support (LPS) increases the live-birth rate (LBR) in women undergoing modified natural cycle (mNC) frozen-thawed embryo transfer (FET). METHODS: In a randomized controlled trial, conducted at a university-affiliated tertiary medical center, a total of 59 patients aged 18-45 years, underwent mNC-FET. FET was performed in mNC following ovulation triggering by hCG. Patients were randomized into two groups; The No-LPS Group included 28 women who did not receive LPS, and the LPS Group included 31 women who received vaginal progesterone for LPS. The main outcome measure was LBR. RESULTS: Baseline demographic and clinical characteristics were comparable between the study groups. The no-LPS group and the LPS group did not differ with regard to clinical pregnancy rate (21.4% vs. 32.3%; respectively, p = .35), LBR (17.9% vs. 19.4%; respectively, p = .88), or spontaneous miscarriage rate (3.6% vs. 12.9%; respectively, p = .35). On multivariate logistic regression analysis, LPS was not associated with LBR after controlling for confounders. CONCLUSION: The results of our study suggest that LPS after mNC-FET does not improve the reproductive outcome, and therefore, might not be necessary.C linicaltrials.gov identifier: NCT01483365.

Ca2+ signaling in mammalian spermatozoa.

Calcium is an essential ion which regulates sperm motility, capacitation and the acrosome reaction (AR), three processes necessary for successful fertilization. The AR enables the spermatozoon to penetrate into the egg. In order to undergo the AR, the spermatozoon must reside in the female reproductive tract for several hours, during which a series of biochemical transformations takes place, collectively called capacitation. An early event in capacitation is relatively small elevation of intracellular Ca2+ (in the nM range) and bicarbonate, which collectively activate the soluble adenylyl cyclase to produce cyclic-AMP; c-AMP activates protein kinase A (PKA), leading to indirect tyrosine phosphorylation of proteins. During capacitation, there is an increase in the membrane-bound phospholipase C (PLC) which is activated prior to the AR by relatively high increase in intracellular Ca2+ (in the µM range). PLC catalyzes the hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (PIP2) to diacylglycerol and inositol-trisphosphate (IP3), leading to activation of protein kinase C (PKC) and the IP3-receptor. PKC activates a Ca2+- channel in the plasma membrane, and IP3 activates the Ca2+- channel in the outer acrosomal membrane, leading to Ca2+ depletion from the acrosome. As a result, the plasma-membrane store-operated Ca2+ channel (SOCC) is activated to increase cytosolic Ca2+ concentration, enabling completion of the acrosome reaction. The hydrolysis of PIP2 by PLC results in the release and activation of PIP2-bound gelsolin, leading to F-actin dispersion, an essential step prior to the AR. Ca2+ is also involved in the regulation of sperm motility. During capacitation, the sperm develops a unique motility pattern called hyper-activated motility (HAM) which is essential for successful fertilization. The main Ca2+-channel that mediates HAM is the sperm-specific CatSper located in the sperm tail.

Actin cytoskeleton and sperm function.

For the acquisition of the ability to fertilize the egg, mammalian spermatozoa should undergo a series of biochemical transformations in the female reproductive tract, collectively called capacitation. The capacitated sperm can undergo the acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize it. One of the main processes in capacitation involves dynamic cytoskeletal remodeling particularly of actin. Actin polymerization occurs during sperm capacitation and the produced F-actin should be depolymerized prior to the acrosomal exocytosis. In the present review, we describe the mechanisms that regulate F-actin formation during sperm capacitation and the F-actin dispersion prior to the acrosomal exocytosis. During sperm capacitation, the actin severing proteins gelsolin and cofilin are inactive and they undergo activation prior to the acrosomal exocytosis.

The role and importance of cofilin in human sperm capacitation and the acrosome reaction.

The spermatozoon is capable of fertilizing an oocyte only after undergoing several biochemical changes in the female reproductive tract, referred to as capacitation. The capacitated spermatozoon interacts with the egg zona pellucida and undergoes the acrosome reaction, which enables its penetration into the egg and fertilization. Actin dynamics play a major role throughout all these processes. Actin polymerization occurs during capacitation, whereas prior to the acrosome reaction, F-actin must undergo depolymerization. In the present study, we describe the presence of the actin-severing protein, cofilin, in human sperm. We examined the function and regulation of cofilin during human sperm capacitation and compared it to gelsolin, an actin-severing protein that was previously investigated by our group. In contrast to gelsolin, we found that cofilin is mainly phosphorylated/inhibited at the beginning of capacitation, and dephosphorylation occurs towards the end of the process. In addition, unlike gelsolin, cofilin phosphorylation is not affected by changing the cellular levels of PIP2. Despite the different regulation of the two proteins, the role of cofilin appears similar to that of gelsolin, and its activation leads to actin depolymerization, inhibition of sperm motility and induction of the acrosome reaction. Moreover, like gelsolin, cofilin translocates from the tail to the head during capacitation. In summary, gelsolin and cofilin play a similar role in F-actin depolymerization prior to the acrosome reaction but their pattern of phosphorylation/inactivation during the capacitation process is different. Thus, for the sperm to achieve high levels of F-actin along the capacitation process, both proteins must be inactivated at different times and, in order to depolymerize F-actin, both must be activated prior to the acrosome reaction.

Regulation of Sperm Capacitation and the Acrosome Reaction by PIP 2 and Actin Modulation.

Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. Actin polymerization occurs during capacitation and prior to the acrosome reaction, fast F-actin breakdown takes place. The increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP 2 ) and its phosphorylation on tyrosine-438 by Src. Activation of gelsolin following its release from PIP 2 is known to cause F-actin breakdown and inhibition of sperm motility, which can be restored by adding PIP 2 to the cells. Reduction of PIP 2 synthesis inhibits actin polymerization and motility, while increasing PIP 2 synthesis enhances these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP 2 and F-actin. During capacitation there was an increase in PIP 2 and F-actin levels in the sperm head and a decrease in the tail. In spermatozoa with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends upon its binding to PIP 2 . Stimulation of phospholipase C, by Ca 2 + -ionophore or by activating the epidermal-growth-factor-receptor, inhibits tyrosine phosphorylation of gelsolin and enhances enzyme activity. In conclusion, these data indicate that the increase of PIP 2 and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result, the decrease of gelsolin in the tail allows the maintenance of high levels of F-actin in this structure, which is essential for the development of HA motility.

Regulation of sperm motility by PIP2(4,5) and actin polymerization.

Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. In previous studies, we demonstrated that the increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP2). Here, we showed for the first time the involvement of PIP2/gelsolin in human sperm motility before and during capacitation. Activation of gelsolin by causing its release from PIP2 inhibited sperm motility, which could be restored by adding PIP2 to the cells. Reduction of PIP2 synthesis inhibited actin polymerization and motility, and increasing PIP2 synthesis enhanced these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP2 and F-actin. During capacitation there was an increase in PIP2 and F-actin levels in the sperm head and a decrease in the tail. In sperm with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends on its binding to PIP2. Activation of phospholipase C by Ca(2+)-ionophore or by activating the epidermal-growth-factor-receptor inhibits tyrosine phosphorylation of gelsolin. In conclusion, the data indicate that the increase of PIP2 and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result the decrease of gelsolin in the tail allows keeping high level of F-actin in the tail, which is essential for the development of HA motility.

Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases.

Mammalian sperm must undergo a series of biochemical and physiological modifications, collectively called capacitation, in the female reproductive tract prior to the acrosome reaction (AR). The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca(2+) during both capacitation and the AR. In the present review, we summarize some of the signaling events that are involved in capacitation. During the capacitation process, phosphatidyl-inositol-3-kinase (PI3K) is phosphorylated/activated via a protein kinase A (PKA)-dependent cascade, and downregulated by protein kinase C α (PKCα). PKCα is active at the beginning of capacitation, resulting in PI3K inactivation. During capacitation, PKCα as well as PP1γ2 is degraded by a PKA-dependent mechanism, allowing the activation of PI3K. The activation of PKA during capacitation depends mainly on cyclic adenosine monophosphate (cAMP) produced by the bicarbonate-dependent soluble adenylyl cyclase. This activation of PKA leads to an increase in actin polymerization, an essential process for the development of hyperactivated motility, which is necessary for successful fertilization. Actin polymerization is mediated by PIP(2) in two ways: first, PIP(2) acts as a cofactor for phospholipase D (PLD) activation, and second, as a molecule that binds and inhibits actin-severing proteins such as gelsolin. Tyrosine phosphorylation of gelsolin during capacitation by Src family kinase (SFK) is also important for its inactivation. Prior to the AR, gelsolin is released from PIP(2) and undergoes dephosphorylation/activation, resulting in fast F-actin depolymerization, leading to the AR.

Role and regulation of sperm gelsolin prior to fertilization.

To acquire fertilization competence, spermatozoa should undergo several biochemical changes in the female reproductive tract, known as capacitation. The capacitated spermatozoon can interact with the egg zona pellucida resulting in the occurrence of the acrosome reaction, a process that allowed its penetration into the egg and fertilization. Sperm capacitation requires actin polymerization, whereas F-actin must disperse prior to the acrosome reaction. Here, we suggest that the actin-severing protein, gelsolin, is inactive during capacitation and is activated prior to the acrosome reaction. The release of bound gelsolin from phosphatidylinositol 4,5-bisphosphate (PIP(2)) by PBP10, a peptide containing the PIP(2)-binding domain of gelsolin, or by activation of phospholipase C, which hydrolyzes PIP(2), caused rapid Ca(2+)-dependent F-actin depolymerization as well as enhanced acrosome reaction. Using immunoprecipitation assays, we showed that the tyrosine kinase SRC and gelsolin coimmunoprecipitate, and activating SRC by adding 8-bromo-cAMP (8-Br-cAMP) enhanced the amount of gelsolin in this precipitate. Moreover, 8-Br-cAMP enhanced tyrosine phosphorylation of gelsolin and its binding to PIP(2(4,5)), both of which inactivated gelsolin, allowing actin polymerization during capacitation. This actin polymerization was blocked by inhibiting the Src family kinases, suggesting that gelsolin is activated under these conditions. These results are further supported by our finding that PBP10 was unable to cause complete F-actin breakdown in the presence of 8-Br-cAMP or vanadate. In conclusion, inactivation of gelsolin during capacitation occurs by its binding to PIP(2) and tyrosine phosphorylation by SRC. The release of gelsolin from PIP(2) together with its dephosphorylation enables gelsolin activation, resulting in the acrosome reaction.