Vitamin D Supplementation in Neonatal and Infant MIS-C Following COVID-19 Infection.

To date, the SARS-CoV-2 pandemic still represents a great clinical challenge worldwide, and effective anti-COVID-19 drugs are limited. For this reason, nutritional supplements have been investigated as adjuvant therapeutic approaches in disease management. Among such supplements, vitamin D has gained great interest, due to its immunomodulatory and anti-inflammatory actions both in adult and pediatric populations. Even if there is conflicting evidence about its prevention and/or mitigation effectiveness in SARS-CoV-2 infection, several studies demonstrated a strict correlation between hypovitaminosis D and disease severity in acute COVID-19 and MIS-C (multisystem inflammatory syndrome in children). This narrative review offers a resume of the state of the art about vitamin D's role in immunity and its clinical use in the context of the current pandemic, specially focusing on pediatric manifestations and MIS-C. It seems biologically reasonable that interventions aimed at normalizing circulating vitamin D levels could be beneficial. To help clinicians in establishing the correct prophylaxis and/or supportive therapy with vitamin D, well-designed and adequately statistically powered clinical trials involving both adult and pediatric populations are needed. Moreover, this review will also discuss the few other nutraceuticals evaluated in this context.

Human papillomavirus infection in women undergoing in-vitro fertilization: effects on embryo development kinetics and live birth rate.

BACKGROUD: Several studies showed that human papillomavirus (HPV) affects male fertility, but its impact on female fertility and in vitro fertilization (IVF) outcome is not yet clear. METHODS: Objective of this observational, prospective, cohort study was to evaluate the prevalence of HPV infection in women candidate to IVF, and the effects of HPV infection on the kinetic of embryonic development and on IVF outcome. A total number of 457 women candidate to IVF were submitted to HR-HPV test; among them, 326 underwent their first IVF cycle and were included in the analysis on IVF results. RESULTS: 8.9% of women candidate to IVF were HPV-positive, HPV16 being the most prevalent genotype. Among the infertility causes, endometriosis was significantly more frequent in HPV-positive than in negative women (31.6% vs. 10.1%; p < 0.01). Granulosa and endometrial cells resulted HPV-positive in 61% and 48% of the women having HPV-positive cervical swab, respectively. Comparing HPV-positive and negative women at their first IVF cycle, no significant difference was observed in the responsiveness to controlled ovarian stimulation (COS) in terms of number and maturity of retrieved oocytes, and of fertilization rate. The mean morphological embryo score was comparable in the two groups; embryos of HPV-positive women showed a quicker development in the early stages, with a significantly shorter interval between the appearance of pronuclei and their fusion. In the following days, embryo kinetic was comparable in the two groups until the early blastocyst stage, when embryos of HPV-positive women became significantly slower than those of HPV-negative women. Overall, these differences did not affect live birth rate/started cycle, that was comparable in HPV-positive and negative women (22.2 and 28.1%, respectively). CONCLUSIONS: (a) the prevalence of HPV infection in women candidate to IVF is similar to that observed in the general female population of the same age range; (b) HPV infection migrates along the female genital apparatus, involving also the endometrium and the ovary, and perhaps participates in the genesis of pelvic endometriosis; (c) HPV slightly affects the developmental kinetic of in vitro-produced embryos, but does not exert an effect on live birth rate.

Cryostorage management of reproductive cells and tissues in ART: status, needs, opportunities and potential new challenges.

Among the wide range of procedures performed by clinical embryologists, the cryopreservation of reproductive cells and tissues represents a fundamental task in the daily routine. Indeed, cryopreservation procedures can be considered a subspecialty of medically assisted reproductive technology (ART), having the same relevance as sperm injection or embryo biopsy for preimplantation genetic testing. However, although a great deal of care has been devoted to optimizing cryopreservation protocols, the same energy has only recently been spent on developing and implementing strategies for the safe and reliable storage and transport of reproductive specimens. Herein, we have summarized the content of the available guidelines, the risks, the needs and the future perspectives regarding the management of cryopreservation biorepositories used in ART.

Retrospective comparison of monochorionic diamniotic twin pregnancies stratified by spontaneous or artificial conception.

OBJECTIVES: To compare outcomes of monochorionic diamniotic (MCDA) twin pregnancies by in vitro fertilization (IVF) vs. spontaneously conceived counterparts. METHODS: Retrospective comparison. Data about MCDA twin pregnancies, conceived spontaneously or by IVF, attending the Twin Pregnancy Care Unit of Sant'Anna Hospital in Turin (Italy) between January 1st 2010 and March 31st 2022, were collected retrospectively. Obstetric, fetal, and neonatal outcomes of MCDA twin pregnancies by IVF were compared to those of spontaneously conceived counterparts. Data were described by univariate and multivariate analysis. RESULTS: 541 MCDA twin pregnancies were included in the study, among which 45 conceived by IVF and 496 spontaneously conceived. Women with IVF twins were older than those who conceived spontaneously (36.7 ± 5.7 vs. 32.1 ± 5.2 years; p<0.001). No significant difference in the prevalence of pregnancy complications between the two groups was found, except for an increased incidence of hypertensive disorders among IVF pregnancies (17.8 vs. 8.5 %; p=0.039), which resulted nonsignificant after adjusting for maternal age and parity (aOR 1.9, 95 % CI 0.8-4.6). Data about 1,046 live born babies (90 conceived by IVF and 956 spontaneously) were also collected: perinatal outcomes did not differ between the two groups. CONCLUSIONS: Our results suggest that MCDA twin pregnancies following IVF are not at increased risk of adverse outcomes compared to spontaneous MCDA twin pregnancies.

Immune Checkpoints in Recurrent Pregnancy Loss: New Insights into a Detrimental and Elusive Disorder.

Recurrent pregnancy loss (RPL) refers to two or more miscarriages before 20 weeks gestation. Its prevalence is 1-2%; its pathogenesis remains unexplained in more than 50% of cases, in which the cause is thought to be abnormal immune activity during placentation leading to a lack of pregnancy-induced immune tolerance. It is unknown whether immune activity is deranged in the endometrium of women with RPL. We studied the gene expression and the quantitative tissue protein levels of three immune checkpoints (CD276, which enhances cytotoxic T-cell activity, cytotoxic T-lymphocyte-associated antigen-4 [CTL-4], which reduces Th1 cytokine production, and lymphocyte activation gene-3 [LAG-3], which shows suppressive activity on Tregs and CD4+ T-cells) in endometrial samples from 27 women with unexplained RPL and in 29 women with dysfunctional uterine bleeding and previous uneventful pregnancies as controls. RNA isolation, real-time PCR, protein isolation, and ELISA were performed. CD276 gene expression and protein tissue levels were significantly lower in the endometrium of the RPL group than in the controls, whereas both CTL-4 and LAG-3 were significantly higher. This difference suggests defective endometrial immune regulation and overactivation of immune response in women with a history of RPL, at least in relation to controls with dysfunctional uterine bleeding and previous normal reproductive history.

Are commercial warming kits interchangeable for vitrified human blastocysts? Further evidence for the adoption of a Universal Warming protocol.

PURPOSE: To study whether a new combination of different warming kits is clinically effective for vitrified human blastocysts. METHODS: This is a longitudinal cohort study analysing two hundred fifty-five blastocysts warming cycles performed between January and October 2018. Embryos were vitrified using only one brand of ready-to-use kits (Kitazato), whereas the warming procedure was performed with three of the most widely used vitrification/warming kits (Kitazato, Sage and Irvine) after patient stratification for oocyte source. The primary endpoint was survival rate, while the secondary endpoints were clinical pregnancy, live birth and miscarriage rates. RESULTS: We observed a comparable survival rate across all groups of 100% (47/47) in KK, 97.6% (49/50) in KS, 97.6% (41/42) in KI, 100% (38/38) in dKK, 100% (35/35) in dKS and 100% (43/43) in dKI. Clinical pregnancy rates were also comparable: 38.3% (18/47) in KK, 49% (24/49) in KS, 56.1% (23/ 41) in KI, 47.4% (18/38) in dKK, 31.4% (11/35) in dKS and 48.8% (21/ 43) in dKI. Finally, live birth rates were 29.8% (14/47) in KK, 36.7% (18/49) in KS, 46.3% (19/41) in KI, 36.8% (14/38) in dKK, 25.7% (9/35) in dKS and 41.9% (18/43) in dKI, showing no significant differences. CONCLUSION: This study confirmed the efficacy of applying a single warming protocol, despite what the "industry" has led us to believe, supporting the idea that it is time to proceed in the cryopreservation field and encouraging embryologists worldwide to come out and reveal that such a procedure is possible and safe.

Second stimulation in the same ovarian cycle: an option to fully-personalize the treatment in poor prognosis patients undergoing PGT-A.

PURPOSE: Our primary objective was to assess whether immediately undergoing a second stimulation in the same ovarian cycle (DuoStim) for advanced-maternal-age and/or poor-ovarian-reserve (AMA/POR) patients obtaining ≤ 3 blastocysts for preimplantation-genetic-testing-for-aneuploidies (PGT-A) is more efficient than the conventional-approach. METHODS: All AMA/POR patients obtaining ≤ 3 blastocysts after conventional-stimulation between 2017 and 2019 were proposed DuoStim, and 143 couples accepted (DuoStim-group) and were matched for the main confounders to 143 couples who did not accept (conventional-group). GnRH-antagonist protocol with recombinant-gonadotrophins and agonist trigger, intra-cytoplasmatic-sperm-injection (ICSI) with ejaculated sperm, PGT-A and vitrified-warmed euploid single-blastocyst-transfer(s) were performed. The primary outcome was the cumulative-live-birth-delivery-rate per intention-to-treat (CLBdR per ITT) within 1 year. If not delivering, the conventional-group had 1 year to undergo another conventional-stimulation. A cost-effectiveness analysis was also conducted. RESULTS: The CLBdR was 10.5% in the conventional-group after the first attempt. Only 12 of the 128 non-pregnant patients returned (165 ± 95 days later; drop-out = 116/128,90.6%), and 3 delivered. Thus, the 1-year CLBdR was 12.6% (N = 18/143). In the DuoStim-group, the CLBdR was 24.5% (N = 35/143; p = 0.01), 2 women delivered twice and 13 patients have other euploid blastocysts after a LB (0 and 2 in the conventional-group). DuoStim resulted in an incremental-cost-effectiveness-ratio of 23,303€. DuoStim was costlier and more effective in 98.7% of the 1000 pseudo-replicates generated through bootstrapping, and the cost-effectiveness acceptability curves unveiled that DuoStim would be more cost-effective than the conventional-approach at a willingness-to-pay threshold of 23,100€. CONCLUSIONS: During PGT-A treatments in AMA/POR women, DuoStim can be suggested in progress to rescue poor blastocyst yields after conventional-stimulation. It might indeed prevent drop-out or further aging between attempts.

Monozygotic twin rate among ART centers: a multicenter analysis of data from 18 Italian units.

PURPOSE: The risk of monozygotic twins (MZTs) is increased in couples undergoing assisted reproductive technology (ART) treatments. Several systematic reviews have investigated the possible determinants linked to ART, but results obtained have not been conclusive. The study aims to investigate whether the incidence of MZT differed among ART centers. METHODS: This is a multicenter retrospective cohort study using the Italian ART National Registry database and involving the centers reporting data from individual ART cycles from 2015 to 2019. To investigate the incidence of MZT, only single embryo transfer cycles were considered. Women who had sex-discordant deliveries were excluded. MZT rate was calculated as the number of multiple pregnancies (more than one gestational sac at first ultrasound) out of the total number of clinical pregnancies. A binomial distribution model was used to determine the 95% CI of the frequency of MZT. RESULTS: Eighteen centers were included, and they provided data on 10,433 pregnancies. The total number of MZT was 162, corresponding to an incidence of 1.5% (95% CI: 1.3-1.8%). The rate of MZT among centers varied between 0% (95% CI: 0.0-25.9%) and 3.2% (95% CI: 1.3-8.1%). All the 95% CIs included 1.5%, rejecting the hypothesis that the MZT rate may significantly differ among centers. CONCLUSIONS: The rate of MZT did not significantly vary among ART centers. Local factors are unlikely to explain the increased rate of MZT in ART pregnancies.

A Novel Xeno-Free Method to Isolate Human Endometrial Mesenchymal Stromal Cells (E-MSCs) in Good Manufacturing Practice (GMP) Conditions.

The cyclic regeneration of human endometrium is guaranteed by the proliferative capacity of endometrial mesenchymal stromal cells (E-MSCs). Due to this, the autologous infusion of E-MSCs has been proposed to support endometrial growth in a wide range of gynecological diseases. We aimed to compare two different endometrial sampling methods, surgical curettage and vacuum aspiration biopsy random assay (VABRA), and to validate a novel xeno-free method to culture human E-MSCs. Six E-MSCs cell samples were isolated after mechanical tissue homogenization and cultured using human platelet lysate. E-MSCs were characterized for the colony formation capacity, proliferative potential, and multilineage differentiation. The expression of mesenchymal and stemness markers were tested by FACS analysis and real-time PCR, respectively. Chromosomal alterations were evaluated by karyotype analysis, whereas tumorigenic capacity and invasiveness were tested by soft agar assay. Both endometrial sampling techniques allowed efficient isolation and expansion of E-MSCs using a xeno-free method, preserving their mesenchymal and stemness phenotype, proliferative potential, and limited multi-lineage differentiation ability during the culture. No chromosomal alterations and invasive/tumorigenic capacity were observed. Herein, we report the first evidence of efficient E-MSCs isolation and culture in Good Manufacturing Practice compliance conditions, suggesting VABRA endometrial sampling as alternative to surgical curettage.

Quinagolide Treatment Reduces Invasive and Angiogenic Properties of Endometrial Mesenchymal Stromal Cells.

Endometrial mesenchymal stromal cells (E-MSCs) extensively contribute to the establishment and progression of endometrial ectopic lesions through formation of the stromal vascular tissue, and support to its growth and vascularization. As E-MSCs lack oestrogen receptors, endometriosis eradication cannot be achieved by hormone-based pharmacological approaches. Quinagolide is a non-ergot-derived dopamine receptor 2 agonist reported to display therapeutic effects in in vivo models of endometriosis. In the present study, we isolated E-MSCs from eutopic endometrial tissue and from ovarian and peritoneal endometriotic lesions, and we tested the effect of quinagolide on their proliferation and matrix invasion ability. Moreover, the effect of quinagolide on E-MSC endothelial differentiation was assessed in an endothelial co-culture model of angiogenesis. E-MSC lines expressed dopamine receptor 2, with higher expression in ectopic than eutopic ones. Quinagolide inhibited the invasive properties of E-MSCs, but not their proliferation, and limited their endothelial differentiation. The abrogation of the observed effects by spiperone, a dopamine receptor antagonist, confirmed specific dopamine receptor activation. At variance, no involvement of VEGFR2 inhibition was observed. Moreover, dopamine receptor 2 activation led to downregulation of AKT and its phosphorylation. Of interest, several effects were more prominent on ectopic E-MSCs with respect to eutopic lines. Together with the reported effects on endometrial and endothelial cells, the observed inhibition of E-MSCs may increase the rationale for quinagolide in endometriosis treatment.

Impaired transcription of human endogenous retroviruses in the sperm with exception of syncytin 1: short communication.

BACKGROUND: Human endogenous retroviruses (HERVs), remnants of ancestral infections, represent 8% of the human genome. HERVs are co-opted for important physiological functions during embryogenesis; however, little is known about their expression in human gametes. We evaluated the transcriptional levels of several retroviral sequences in human spermatozoa. METHODS AND RESULTS: We assessed, through a Real-Time PCR assay, the transcription levels of the pol genes of HERV-H, -K and -W families and of env genes of syncytin (Syn)1 and Syn2 in the spermatozoa from 8 normospermic subjects. The entity and distribution of their expressions were compared to values found in white blood cells (WBCs) from 16 healthy volunteers. The level of HERV transcripts was significantly lower in spermatozoa than in WBCs for HERV-H-pol, HERV-K-pol, HERV-W-pol, and Syn2.In contrast, the level of expression of Syn1 in the sperm was similar to that found in WBCs and it was significantly higher than the mRNA concentrations of other HERV genes in spermatozoa. CONCLUSIONS: Our findings show, for the first time, the presence of several retroviral mRNAs in the sperm, although in low amounts. The higher concentration of Syn1 suggests that it could play a key role in the fusion process between gametes during fertilization and, perhaps, be involved in embryo development. Further studies could clarify whether aberrant HERV expressions, in particular of Syn1, negatively affect fertilization and embryo growth and whether sperm manipulation procedures, such as cryopreservation, may potentially influence HERV transcription in the human male gamete.

A prospective randomized trial comparing corifollitropin-α late-start (day 4) versus standard administration (day 2) in expected poor, normal, and high responders undergoing controlled ovarian stimulation for IVF.

OBJECTIVE: To assess whether corifollitropin-α (CFα) late-start administration (day 4) and standard administration (day 2) can obtain similar oocyte yield and live birth rate. STUDY DESIGN: A randomized controlled trial. SETTING: University Hospital IVF Unit. PATIENTS: One hundred thirteen women undergoing IVF. INTERVENTIONS: Patients distributed in three subgroups (expected poor, normal, or high responders to FSH) were randomized into two treatment arms: (a) CFα late-start: CFα on day 4 + GnRH antagonist from day 8 + (when needed) recFSH from day 11; (b) CFα standard start: CFα on day 2 + GnRH antagonist from day 6 + (when needed) recFSH from day 9. IVF or ICSI was performed as indicated. RESULTS: Considering the whole study group, the late-start regimen obtained comparable oocyte yield (8.9 ± 5.6 vs. 8.8 ± 6.2; p = n.s.), cPR/started cycle (25% vs. 31.6%, p = n.s.), and cumulative live birth rate (LBR)/ovum pickup (OPU) (29.2% vs. 37.7%, p = n.s.) than the standard regimen. The outcome of the two regimens was comparable in the two subgroups of high and normal responders. Differently, in poor responders, oocyte yield was similar, but LBR/OPU was significantly lower with late-start CFα administration that caused 40% cancellation rate due to monofollicular response. ROC curves showed that the threshold AMH levels associated with cycle cancellation were 0.6 ng/ml for late-start regimen and 0.2 ng/ml for standard regimen. CONCLUSION: CFα may be administered on either day 2 or day 4 to patients with expected high or normal response to FSH without compromising oocyte yield and/or live birth rate. Differently, late-start administration is not advisable for expected poor responders with AMH ≤ 0.6 ng/ml. TRIAL REGISTRATION: NCT03816670.

Is guided, targeted information about the risks of twin pregnancy able to increase the acceptance of single embryo transfer among IVF couples? A prospective study.

OBJECTIVE: To assess whether receiving information about twin pregnancy in the form of oral presentation given by a physician could affect the acceptance of single embryo transfer (SET) by couples undergoing IVF. STUDY DESIGN: Prospective interventional study. SETTING: University hospital IVF unit. PATIENTS: One hundred and forty patients (70 couples) undergoing IVF. INTERVENTIONS: A questionnaire to measure patients' emotions about twin pregnancy was administered to IVF patients just before and immediately after attending a slide presentation in which the risks of twin pregnancy were explained. Patients scored (1 to 6) ten adjectives linked either to positive or negative emotions; scores before and after presentation were compared. The patients' preference between double embryo transfer (DET) and SET was also registered before and after the presentation. RESULTS: The presentation about twin pregnancy caused a significant (p < 0.001) shift of the score distribution toward lower values for positive adjectives referred to twin pregnancy and higher values for negative adjectives. Information impacted similarly on women and men. Despite the relevant change in the emotional attitude, after presentation, 45.7% of women and 48.6% of men were still favorable to DET, whereas 24.3% of women and 37.1% of men preferred SET. CONCLUSIONS: Oral information on the risks of twin pregnancy can affect the emotional attitude of patients toward twin pregnancy, but the wish of getting pregnant after fresh embryo transfer overcomes all rational consideration, and the majority of patients still prefer DET.

Correction to: A prospective randomized trial comparing corifollitropin-α late-start (day 4) versus standard administration (day 2) in expected poor, normal, and high responders undergoing controlled ovarian stimulation for IVF.

The original article unfortunately has a missing acknowledgement.

Controlled ovarian stimulation and progesterone supplementation affect vaginal and endometrial microbiota in IVF cycles: a pilot study.

PURPOSE: Does controlled ovarian stimulation (COS) and progesterone (P) luteal supplementation modify the vaginal and endometrial microbiota of women undergoing in vitro fertilization? METHODS: Fifteen women underwent microbiota analysis at two time points: during a mock transfer performed in the luteal phase of the cycle preceding COS, and at the time of fresh embryo transfer (ET). A vaginal swab and the distal extremity of the ET catheter tip were analyzed using next-generation 16SrRNA gene sequencing. Heterogeneity of the bacterial microbiota was assessed according to both the Bray-Curtis similarity index and the Shannon diversity index. RESULTS: Lactobacillus was the most prevalent genus in the vaginal samples, although its relative proportion was reduced by COS plus P supplementation (71.5 ± 40.6% vs. 61.1 ± 44.2%). In the vagina, an increase in pathogenic species was observed, involving Prevotella (3.5 ± 8.9% vs. 12.0 ± 19.4%), and Escherichia coli-Shigella spp. (1.4 ± 5.6% vs. 2.0 ± 7.8%). In the endometrium, the proportion of Lactobacilli slightly decreased (27.4 ± 34.5% vs. 25.0 ± 29.9%); differently, both Prevotella and Atopobium increased (3.4 ± 9.5% vs. 4.7 ± 7.4% and 0.7 ± 1.5% vs. 5.8 ± 12.0%). In both sites, biodiversity was greater after COS (p < 0.05), particularly in the endometrial microbiota, as confirmed by Bray-Curtis analysis of the phylogenetic distance among bacteria genera. Bray-Curtis analysis confirmed significant differences also for the paired endometrium-vagina samples at each time point. CONCLUSIONS: Our findings suggest that COS and P supplementation significantly change the composition of vaginal and endometrial microbiota. The greater instability could affect both endometrial receptivity and placentation. If our findings are confirmed, they may provide a further reason to encourage the freeze-all strategy.

Morphokinetic analysis of cleavage stage embryos and assessment of specific gene expression in cumulus cells independently predict human embryo development to expanded blastocyst: a preliminary study.

To assess whether morphokinetic features at the cleavage stage together with specific gene expression in cumulus cells (CCs) may be used to predict whether human embryos are able to achieve the expanded blastocyst stage on day 5. Eighty-one embryos were cultured using the Geri plus® time-lapse system. Twenty-seven embryos progressing to the expanded blastocyst stage (BL group) were compared with thirty-five embryos showing developmental arrest (AR group) and nineteen reaching the stage of early or not fully expanded blastocyst (nBL group). The analyzed morphokinetic variables were pronuclear appearance (tPNa), pronuclear fading (tPNf), and completion of cleavage to two, three, four, and eight cells (t2, t3, t4, and t8). CCs were analyzed by RT-qPCR for bone morphogenetic protein 15 (BMP15), cytochrome c oxidase subunit II (COXII), ATP synthase subunit 6 (MT-ATP6), connexin 43 (Cx43), and heme oxygenase-1 (HO-1). Embryos of BL group showed a significantly faster kinetic. BMP15, COXII, and MT-ATP6 mRNA expression was significantly higher in CCs of BL group embryos, whereas Cx43 and HO-1 mRNA levels were higher in AR group. Kinetic parameters and gene expression were not significantly different between either the BL and nBL groups or the AR and nBL groups. ROC curves showed that the most predictive cut-offs were t2 < 26.25 for morphokinetics and COXII > 0.3 for gene expression. Multivariable logistic regression analysis showed that morphokinetic variables and gene expression were both valuable, independent predictors of embryo development to expanded blastocyst. Our results suggest the possibility of developing integrated prediction models for early embryo selection at the cleavage stage.

Human Ovarian Cortex biobanking: A Fascinating Resource for Fertility Preservation in Cancer.

Novel anti-cancer treatments have improved the survival rates of female young patients, reopening pregnancy issues for female cancer survivors affected by the tumor treatment-related infertility. This condition occurs in approximately one third of women of fertile age and is mainly dependent on gonadotoxic protocols, including radiation treatments. Besides routine procedures such as the hormonal induction of follicular growth and subsequent cryopreservation of oocytes or embryos, the ovarian protection by gonadotropin-releasing hormone (GnRH) agonists during chemotherapy as well as even gonadal shielding during radiotherapy, other innovative techniques are available today and need to be optimized to support their introduction into the clinical practice. These novel methods are hormone stimulation-free and include the ovarian cortex cryopreservation before anti-cancer treatments and its subsequent autologous reimplantation and a regenerative medicine approach using oocytes derived in vitro from ovarian stem cells (OSCs). For both procedures, the major benefit is related to the prompt recruitment and processing of the ovarian cortex fragments before gonadotoxic treatments. However, while the functional competence of oocytes within the cryopreserved cortex is not assessable, the in vitro maturation of OSCs to oocytes, allows to select the most competent eggs to be cryopreserved for fertility restoration.

Inferring biallelism of two FSH receptor mutations associated with spontaneous ovarian hyperstimulation syndrome by evaluating FSH, LH and HCG cross-activity.

RESEARCH QUESTION: What is the cumulative effect of two follicle-stimulating hormone receptor (FSHR) mutations in spontaneous ovarian hyperstimulation syndrome (sOHSS) pathogenesis? Are these mutations in the mono- or biallelic state? DESIGN: Two FSHR mutations were found in a pregnant patient affected by sOHSS with no predisposing conditions. While the p.Asn106His mutation is novel, the p.Ser128Tyr mutation has been associated with sOHSS previously. The patient's FSHR gene was analysed by Sanger sequencing, and FSHR cDNAs carrying a single or both point mutations were created by mutagenesis in vitro. cAMP activation by recombinant FSH, luteinizing hormone (LH), human chorionic gonadotropin (HCG) and thyroid-stimulating hormone (TSH) was evaluated in transfected HEK293 cells by bioluminescence resonance energy transfer. RESULTS: All mutations decreased the 50% effective concentration of FSH calculated for cAMP (P < 0.05, n = 6), resulting in two- to 10-fold lower ligand potency. TSH failed to induce an FSHR-mediated increase in intracellular cAMP, while LH was approximately four-fold more potent than HCG in p.Ser128Tyr FSHR-expressing HEK293 cells despite lower cAMP plateau levels (P < 0.05, n = 5). The p.Ser128Tyr FSHR mutation was found to be responsible for an LH-/HCG-induced increase in cAMP when it was in the biallelic heterozygous state with p.Asn106His, but no increase in cAMP was induced in the monoallelic state. CONCLUSION: In-vitro data support that, in pregnant patients with sOHSS, the two FSHR mutations have an opposing effect on the pathogenesis of sOHSS and are in the biallelic heterozygous form, allowing HCG to induce a p.Ser128Tyr FSHR-mediated increase in cAMP.

Oocyte polarized light microscopy, assay of specific follicular fluid metabolites, and gene expression in cumulus cells as different approaches to predict fertilization efficiency after ICSI.

BACKGROUND: The complex relationship between oocyte morphology, specific follicular fluid metabolites, gene expression in cumulus granulosa cells, and oocyte competence toward fertilization and embryo development still needs further clarification. METHODS: Forty-six oocytes retrieved from the largest pre-ovulatory follicle of patients undergoing intra-cytoplasmic sperm injection (ICSI) were considered assessing: (a) oocyte morphological characteristics at polarized light microscopy (PLM), (b) specific follicular fluid (FF) metabolites previously suggested to influence oocyte competence (AMH, markers of redox status and of cytotoxicity), (c) transcription of AMH and AMH type II receptor genes in cumulus cells. Data were analyzed using mono-parametric tests and multivariable logistic analysis in order to correlate morphological and biochemical data with fertilization. RESULTS: Comparing normally fertilized oocytes (n = 29, F group) with unfertilized (n = 17, nF group) we observed that: (a) the meiotic spindle area and major axis were significantly higher in nF group and in fertilized oocytes undergoing an early embryo development arrest; (b) AMH level in FF was comparable in F and nF groups; (c) the FF of nF group contained significantly higher levels of cytotoxicity (lactate dehydrogenase) and oxidative stress (Cu,Zn-superoxide dismutase, catalase, 4-hydroxynonenal-protein conjugates) markers; (d) cumulus cells of nF group showed significantly higher AMH receptor type II gene expression. CONCLUSIONS: Taken together, these observations suggest that an excessive cytotoxicity level can alter AMH signal transduction within cumulus cells, in turn leading to partial inhibition of aromatase activity, altered cytoplasmic maturation and increased oxidative stress, factors able to impair oocyte fertilization competence and embryo growth.

Empty follicle syndrome revisited: definition, incidence, aetiology, early diagnosis and treatment.

In this review, the definition, incidence and possible causes of empty follicle syndrome (EFS), including molecular mechanisms that may underlie the syndrome, are discussed, along with prevention and treatment options. EFS is the complete failure to retrieve oocytes after ovarian stimulation, despite apparently normal follicle development and adequate follicular steroidogenesis. Two variants of EFS have been described: the 'genuine' form (gEFS), which occurs in the presence of adequate circulating HCG levels at the time of oocyte aspiration, and the 'false' form (f-EFS), which is associated with circulating HCG below a critical threshold. Heterogeneous HCG concentration thresholds, however, have been used to define gEFS, and to date no standardization exist. The situation is unclear when GnRH-analogues are used for ovulation trigger, as the threshold circulating LH and progesterone levels used to define EFS as 'genuine' are not established. The cause of fEFS has been clearly identified as an error in HCG administration at the time of ovulation trigger; in contrast, the cause of gEFS is still unclear, although some pathogenetic hypotheses have been proposed. Optimal treatment and prognosis of these patients are still poorly understood. Large, systematic multi-centre studies are needed to increase the understanding of EFS.