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To evaluate the association between progesterone (P) level on the day of trigger and time to blastulation in IVF cycles.This was a retrospective cohort study with autologous IVF cycles performed at our Institution from January 2019 to December 2021. A total of 1109 IVF cycles were included. The primary outcome was to compare time to blastulation in terms of percentage of expanded (grade 3) blastocysts on day 5 according to progesterone level at trigger.A total of 3517 blastocysts were analyzed. After dividing progesterone level in quartiles (Q1, P 0.50 ng/ml; Q2 0.50 ng/ml ≤ P ≤ 0.78 ng/ml; Q3, 0.79 ng/ml ≤ P ≤ 1.15 ng/ml; Q4, P 1.15 ng/ml), we observed a delay in blastocyst development according to the increasing level of progesterone at trigger (analysis by rank, P-value = 0.01). After adjusting for confounding factors at the multivariate analysis, the percentage of day 5 blastocysts was reduced for Q3 (- 13.8%, 95% CI from - 20.5 to - 7.0%, p 0.001) and Q4 (- 7.7%, 95% CI from - 15.5 to 0.0%, p = 0.05) compared to Q1 (reference).Progesterone levels on day of trigger correlate to the percentage of expanded (grade 3) blastocysts on day 5 and a delayed blastocyst development day 5 is expected for high progesterone levels.

Purpose Infertility is increasing worldwide, and many couples seek IVF. Clinical management and laboratory work are fundamental in the IVF journey. Therefore, the definition of reliable key performance indicators (KPIs) based on clinical and laboratory parameters, is essential for internal quality control (IQC). Laboratory performance indicators have been identified and a first attempt to also determine clinical ones has been recently published. However, more detailed indicators are required. Methods An Italian group of experts in Reproductive Medicine from both public and private clinics on behalf of SIFES-MR and SIERR was established to define IVF indicators to monitor clinical performance. Results The working group built a consensus on a list of KPIs, performance indicators (PIs) and recommendation indicators (RIs). When deemed necessary, the reference population was stratified by woman age, response to ovarian stimulation and adoption of preimplantation genetic testing for aneuploidies (PGT-A). Each indicator was scored with a value from 1 to 5 and a weighted average formula – considering all the suggested parameters—was defined. This formula generates a center performance score, indicating low, average, good, or excellent performance. Conclusion This study is intended to provide KPIs, PIs and RIs that encompass several essential aspects of a modern IVF clinic, including quality control and constant monitoring of clinical and embryological features. These indicators could be used to assess the quality of each center with the aim of improving efficacy and efficiency in IVF.

Human embryos are very frequently affected by maternally inherited aneuploidies, which in the vast majority of cases determine developmental failure at pre- or post-implantation stages. However, recent evidence, generated by the alliance between diverse technologies now routinely employed in the IVF laboratory, has revealed a broader, more complex scenario. Aberrant patterns occurring at the cellular or molecular level can impact at multiple stages of the trajectory of development to blastocyst. In this context, fertilization is an extremely delicate phase, as it marks the transition between gametic and embryonic life. Centrosomes, essential for mitosis, are assembled ex novo from components of both parents. Very large and initially distant nuclei (the pronuclei) are brought together and positioned centrally. The overall cell arrangement is converted from being asymmetric to symmetric. The maternal and paternal chromosome sets, initially separate and scattered within their respective pronuclei, become clustered where the pronuclei juxtapose, to facilitate their assembly in the mitotic spindle. The meiotic spindle is replaced by a segregation machinery that may form as a transient or persistent dual mitotic spindle. Maternal proteins assist the decay of maternal mRNAs to allow the translation of newly synthesized zygotic transcripts. The diversity and complexity of these events, regulated in a precise temporal order and occurring in narrow time windows, make fertilization a highly error-prone process. As a consequence, at the first mitotic division, cellular or genomic integrity may be lost, with fatal consequences for embryonic development.

Research question What is the intra- and inter-centre reliability in embryo grading performed according to the Istanbul Consensus across several IVF clinics? Design Forty Day 3 embryos and 40 blastocysts were photographed on three focal planes. Senior and junior embryologists from 65 clinics were invited to grade them according to the Istanbul Consensus (Study Phase I). All participants then attended interactive training where a panel of experts graded the same embryos (Study Phase II). Finally, a second set of pictures was sent to both embryologists and experts for a blinded evaluation (Study Phase III). Intra-centre reliability was reported for Study Phase I as Cohen's kappa between senior and junior embryologists; inter-centre reliability was instead calculated between senior/junior embryologists and experts in Study Phase I versus III to outline improvements after training (i.e. upgrade of Cohen's kappa category according to Landis and Koch). Results Thirty-six embryologists from 18 centres participated (28% participation rate). The intra-centre reliability was (i) substantial (0.63) for blastomere symmetry (range –0.02 to 1.0), (ii) substantial (0.72) for fragmentation (range 0.29–1.0), (iii) substantial (0.66) for blastocyst expansion (range 0.19–1.0), (iv) moderate (0.59) for inner cell mass quality (range 0.07–0.92), (v) moderate (0.56) for trophectoderm quality (range 0.01–0.97). The inter-centre reliability showed an overall improvement from Study Phase I to III, from fair (0.21–0.4) to moderate (0.41–0.6) for all parameters under analysis, except for blastomere fragmentation among senior embryologists, which was already moderate before training. Conclusions Intra-centre reliability was generally moderate/substantial, while inter-centre reliability was just fair. The interactive training improved it to moderate, hence this workflow was deemed helpful. The establishment of external quality assessment services (e.g. UK NEQAS) and the avant-garde of artificial intelligence might further improve the reliability of this key practice for embryo selection.

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, we propose that a concomitant laparoscopic oocyte retrieval for cryopreservation is performed. Oocyte retrieval is pursued at the beginning of the laparoscopy. Right and left 5 mm trocars are positioned, through which a 17 G ovum aspiration needle is used for puncture of the right and left ovaries, respectively. To facilitate exposure of the follicles, the ovaries are mobilized and held with laparoscopic forceps. When aspirating multiple follicles near each other, the needle tip is retained in the ovary to reduce the number of times that the ovarian cortex is transfixed and due to the inherent risk of bleeding. Subsequent steps are unchanged compared to the Davydov laparoscopic modified technique for vaginoplasty. Prior to surgery, controlled ovarian stimulation is performed with a gonadotropin hormone-releasing hormone (Gn-RH) antagonist protocol, and the concomitant procedure of oocyte retrieval and vaginoplasty is scheduled 36 h after the final follicular maturation trigger. Follicular fluid is collected in the same 10 mL sterile tubes used during transvaginal oocyte retrieval and transferred in a warming block (37 °C) to the assisted reproduction laboratory, where mature (metaphase II) oocytes are vitrified. In this case, a series of 23 women with MRKH, oocytes were successfully retrieved and cryopreserved in all patients; vaginoplasty was subsequently conducted without modifications, and the inpatient and outpatient postoperative care (day of urinary catheter removal, day of hospital discharge, dilator use, and comfort at follow-up) remained unaffected. One postoperative complication occurred in one patient (fever developing on day 5 post surgery and intraperitoneal fluid detection on transabdominal ultrasound) and resolved after conservative treatment. Rather than performing surgical vaginoplasty and delaying oocyte retrieval in MRKH patients, this approach combines both procedures in a single laparoscopy, thereby minimizing surgical invasiveness and anesthesiologic risks.

Protein sources used as supplements of IVF culture media are known to have several implications for the function and stability of embryo culture environment. In fact, they i) transport biologically active molecules ii) chelate heavy metals, iii) regulate media pH, iii) scavenge reactive oxygen species (ROS) and iv) attenuate osmotic stress to which cells are exposed in sub-optimal culture conditions. Instead, their specific relevance to the formulation of cryopreservation solutions used for gamete and embryo cryopreservation remains uncertain. In the present work, we tested the hypothesis that different protein supplements present in cryopreservation solutions, serum or plasma protein solution (PPS), or different concentrations of the same supplement (serum), are associated with different types and/or magnitude of cryopreservation-derived cell damage. To this end, using cryopreservation solutions containing serum or PPS, donated supernumerary human mature oocytes were frozen-thawed by slow freezing and compared with fresh controls. Ultrastructural markers of oocyte quality were adopted as objective measure to assess possible damage from cryopreservation. The study results indicate that the adoption of serum minimises cell damage induced by cryopreservation. Indeed, typical hallmarks of cryodamage in human oocytes, i.e. loss of cortical granules, zona pellucida hardening and above all vacuolization, were largely reduced in oocytes cryopreserved with solutions containing serum, especially if used a higher concentration. This suggest that oocyte cryopreservation still has significant margins of improvement that may derive also from composition of cryopreservation media.

A recent study by Munné et al. portrayed a protocol to retrieve in vivo produced blastocysts after IUI and uterine lavage for preimplantation genetic testing (PGT) purposes. The authors claimed this protocol might represent a reasonable future perspective for patients who do not want to undergo IVF, but still want to be informed about their embryos’ genetic/chromosomal defects. Although the intent of making PGT available also to patients who cannot or do not need to undergo IVF is respectable, the value of this study is undermined by severe technical and ethical issues. Munné and colleagues’ paper was discussed within the executive committee (i.e., president and vice-president of the society, director and vice-director of the scientific committee, secretariat, and counselors), the special interest group in reproductive genetics, the scientific committee, and the collegio dei probiviri of the Italian Society of Embryology, Reproduction and Research (SIERR). The points raised from this discussion are summarized in this opinion paper.

Study question Are the embryologists across several IVF clinics concordant when evaluating embryo morphology? Summary answer Embryo morphological grading is sufficiently consistent among embryologists from the same center, while an interactive training was essential to improve its accuracy across several clinics. What is known already Embryo morphology, mostly at the blastocyst stage, is the strongest non-invasive embryological feature that associates with implantation potential. This association is confirmed also when euploid blastocysts are transferred. At present, several embryo grading schemes exist but is still unclear which is the most effective among them. Moreover, many IVF clinics adopt internal embryo grading scores, further limiting the transferability of this crucial prognostic information across different laboratories. With the aim of assessing the level of concordance in embryo grading within and between IVF clinics, the Italian Society of Embryology, Reproduction and Research (SIERR) conceived this study. Study design, size, duration We photographed 40 cleavage-stage and 40 blastocyst-stage embryos (3 focal-planes=240 photos). Two embryologists (senior and junior) from 65 Italian IVF clinics were invited to grade them. Their evaluations were blindly collected as Phase-I (January2020-March2020). Phase-II consisted of an interactive-training on Google-Classroom during which 6 selected experts found a Consensus on the morphological evaluation of the 80 embryos (April2020). As Phase III (May2020-July2020), a second set of 240 pictures was sent to senior participants and experts. Participants/materials, setting, methods Eighteen centers agreed to participate, and 36 embryologists were included. The embryo grading scheme adopted was the Alpha-ESHRE Istanbul Consensus (parameters: cleavage-stage blastomeres’ symmetry and fragmentation, blastocyst’s expansion, inner-cell-mass and trophectoderm quality), conventionally used in 50% of the centers (N = 9/18). The concordance within (junior versus senior) and between (senior versus experts) centers was calculated through the Cohen’s-k. The concordance between centers was compared before and after the interactive training on the two sets of pictures. Main results and the role of chance The centers and embryologists included were representative of the Italian IVF scenario: oocyte-retrievals per year:711±636,range100–2200; cycles with cleavage-stage embryo-transfer:322±339,0–1300; cycles with blastocyst-stage embryo-transfer:390±403,0–1100; operators per center:5.6±4.0,2–13; senior embryologists’ experience:14.8±7.4yr,7–30; junior embryologists’ experience:2.7±0.6yr,1–3. The intra-center concordance was (i)for blastomeres’ symmetry 82±15% (38–100%), k 0.59±0.27 (0.02–1), (ii)for blastomeres’ fragmentation 88±9% (65–100%), k 0.71±0.2 (0.29–1), (iii)for blastocysts’ expansion 80±16% (48–100%), k 0.66±0.27 (0.19–1), (iv)for inner-cell-mass quality 73±16% (35–95%), k 0.58±0.24 (0.07–0.92), (v)for trophectoderm quality 71±19% (38–95%), k 0.54±0.32 (0.01–0.97). Linear regressions showed no association of centers’ and embryologists’ characteristics with all k-values. Among clinics with the highest mean number of cycles per year and intra-center concordance, we selected 6 experts for the interactive-training. We then calculated the inter-center concordance as the agreement rate between senior embryologists and the experts for phase-I and phase-III: (i)for blastomeres’ symmetry 67±15% (30–85%) and 73±17% (15–90%;Wilcoxon-signed-ranks-test=0.06), k 0.33±0.22 (–0.29–0.58) and 0.42±0.33 (–0.56–0.77); (ii)for blastomeres’ fragmentation 81±17% (23–95%) and 83±14% (50–95%;Wilcoxon-signed-ranks-test=0.8), k 0.54±0.22 (–0.05–0.84) and 0.55±0.22 (0.17–0.81); (iii)for blastocysts’ expansion 59±16% (35–85%) and 67±17% (23–90%;Wilcoxon-signed-ranks-test=0.04), k 0.35±0.20 (0.06–0.73) and 0.44±0.22 (–0.10–0.7); (iv)for inner-cell-mass quality 60±14% (33–80%) and 69±11% (48–85%;Wilcoxon-signed-ranks-test=0.02), k 0.40±0.20 (0.01–0.69) and 0.51±0.18 (0.18–0.77); (v)for trophectoderm quality 55±12% (23–70%) and 63±10% 48–78%;Wilcoxon-signed-ranks-test Limitations, reasons for caution Only 28% (N = 18/65) of the Italian IVF centers invited to participate responded to the survey. The conventional adoption of grading schemes other than Istanbul-Consensus by 50% of the embryologists might have biased their evaluation. The experts were not fully-concordant in grading 13.8% of the embryos (N = 22/160), which required active discussions. Wider implications of the findings: Blastocyst-grading concordance was significantly improved after the training phase. Therefore, interactive consensus meetings and training platforms are keenly needed to standardize this practice across the centers. The “avant-garde” of artificial intelligence applied to embryo image analysis might help overcoming this issue in the future. Trial registration number N.A.

Introduction: In Italy, the transport of cryopreserved biological material is controlled by several Decrees (Legislative Decree No. 191/2007 and No. 16/2010 and Health Ministry's Decree of October 10, 2012). Given the nature of their applications, the transport of reproductive cells has peculiar quality and safety requirements that must be applied universally, minimizing the chance of error. To standardize the cross-border shipping procedure to meet the quality, traceability, and safety criteria for cells and tissues, it is appropriate to establish a unified process using the same tools, forms, and communication channels. Methods: A working group has been created by SIERR. This "FOCUS Group" was constituted by representatives from Italian-assisted reproductive technology centers and sperm banks who worked together to define joint procedural steps and create specific forms to support the movement of cryopreserved samples. Results: The FOCUS Group identified the critical steps in the communication procedures between Italian centers and created the related forms: patient authorization, request from the recipient center, critical checks carried out by both sending and recipient centers, start of samples transfer, collection, transport and taking responsibility of the biological material, acknowledgment of samples arrival, and acknowledgement of any adverse event that occurred. Discussion: Indications on shipping between tissue institutions and legal responsibilities are important points and a working protocol with shared transport forms has been defined. Standard Operating Procedures are necessary in light of the increasingly widespread movement of biological samples between the various countries, and represent a valid means of support for the patients who could have a higher awareness of safety and traceability during each stage of gamete transport.

A clear definition of developmentally incompetent preimplantation embryo (DIPE) in literature is still missing, while several scientific societies are discussing this challenging topic. From both a clinical and scientific perspective, the identification of embryos unfit for reproductive purpose is crucial. This aim should be pursued in light of all diagnostic technologies for embryo evaluation, encompassing also genetic analyses, of recent implementation in IVF. The Italian context is characterized by an unusual scenario: embryos can be discarded only if not viable and cannot be used for research purposes either. Therefore, thousands of embryos, diagnosed as affected and/or aneuploid as resulting from preimplantation genetic testing (PGT) and clinically not utilizable, are cryopreserved and stored indefinitely, with important psychological, legal, and financial implications. With the aim of updating the definition of DIPE, also on the basis of the embryo genetic status, the Italian Society of Embryology, Reproduction and Research (SIERR) and the Italian Society of Human Genetic (SIGU) reviewed the literature on this topic, found a consensus, and produced a list of relevant criteria.

A realistic picture of our world shows that it is heavily polluted everywhere. Coastal regions and oceans are polluted by farm fertilizer, manure runoff, sewage and industrial discharges, and large isles of waste plastic are floating around, impacting sea life. Terrestrial ecosystems are contaminated by heavy metals and organic chemicals that can be taken up by and accumulate in crop plants, and water tables are heavily contaminated by untreated industrial discharges. As deadly particulates can drift far, poor air quality has become a significant global problem and one that is not exclusive to major industrialized cities. The consequences are a dramatic impairment of our ecosystem and biodiversity and increases in degenerative or man-made diseases. In this respect, it has been demonstrated that environmental pollution impairs fertility in all mammalian species. The worst consequences are observed for females since the number of germ cells present in the ovary is fixed during fetal life, and the cells are not renewable. This means that any pollutant affecting hormonal homeostasis and/or the reproductive apparatus inevitably harms reproductive performance. This decline will have important social and economic consequences that can no longer be overlooked.

Background: folliculogenesis is a strictly regulated process that may be affected by endocrine disrupting chemicals (EDCs) through sometimes not so clear molecular mechanisms. Methods: we conducted a multicentric observational study involving six fertility centers across Italy, prospectively recruiting 122 women attending a fertility treatment. Recruited women had age ≤42 years, and normal ovarian reserve. Blood and follicular fluid samples were taken for EDCs measurement using liquid chromatography tandem mass spectrometry and each woman completed an epidemiological questionnaire. Results: The main EDCs found were monobutyl phthalate (MBP) (median blood: 8.96 ng/mL, follicular fluid 6.43 ng/mL), monoethylhexyl phthalate (MEHP) (median blood: 9.16 ng/mL, follicular fluid 7.68 ng/mL) and bisphenol A (BPA) (median blood: 1.89 ng/mL, follicular fluid 1.86 ng/mL). We found that serum MBP concentration was significantly associated with the considered area (p < 0.001, adj. mean: 7.61 ng/mL, 14.40 ng/mL, 13.56 ng/mL; Area 1: Milan–Turin, Area 2: Rome–Naples; Area 3: Catania–Bari, respectively) but negatively with home plastic food packaging (p = 0.004). Follicular MBP was associated with irregular cycles (p = 0.019). No association was detected between EDCs and eating habits and other clinical and epidemiological features. Conclusions: This study represents the first Italian biomonitoring of plastic EDCs in follicular fluid, laying the basis for future prospective evaluation on oocyte quality before assisted reproduction techniques (ART).

Phthalates and bisphenol A interfere with the synthesis, secretion, transport, binding, metabolism, and excretion of endogenous hormones and, for this reason, are classified as endocrine disruptors. We are here presenting an analytical method for the simultaneous detection of six phthalates metabolites and bisphenol A in different biological fluids (urine, serum and follifular fluid) by liquid chromatography coupled to tandem mass spectrometry. The quantification was carried out in negative electrospray ionization mode using selected reaction monitoring as acquisition mode. Different extraction protocols, using either solid phase or liquid/liquid extraction, were comparatively evaluated to optimize the sample preparation procedure. Solid-phase extraction was chosen as it ensured the best recovery and overall sensitivity. The method was successfully validated: recovery varying in the range 71 ± 2%–107 ± 6% depending on the target analyte and the matrix considered, intra-assay and inter-assay precision ≤ 12% for follicular fluid, ≤11% for serum and ≤ 10% for urine and accuracy ≤ 115% for follicular fluid, ≤113% for serum ≤ 115% for urine , linearity with R2 > 0.99, with the exception of MEP (recovery 64 ± 8%, intra-assay precision ≤ 20%, inter-assay precision ≤ 16% for follicular fluid). The actual applicability of the method developed and validated in this study was assessed by the analysis of real samples, including 10 specimens of follicular fluid, serum and urine samples, that showed the presence of phthalates metabolites and Bisphenol A, and confirming that the newly developed method can be applied in the routine clinical laboratory for the identification and quantitation of these endocrine-disrupting chemicals.

Women affected by ovarian pathologies or with cancer can usually preserve fertility by egg/embryo freezing. When oocyte retrieval is not feasible, the only option available is ovarian tissue cryopreservation and transplantation. The culture of follicles isolated from fresh or cryopreserved ovaries is considered still experimental, although this procedure is considered safer, because the risk of unintentional spreading of cancer cells eventually present in cryopreserved tissue is avoided. Animal and human small follicles can be cultured in vitro, but standardized protocols able to produce in vitro grown oocytes with the same developmental capacity of in vivo grown oocytes are not available yet. In fact, the different sizes of follicles and oocytes, the hormonal differences existing between mono- (e.g., human, goat, cow, and sheep) and poly-ovulatory (rodents and pig) species, and the incomplete identification of the mechanisms regulating the oocyte–follicle and follicle–ovary interrelationships affect the outcome of in vitro culture. From all these attempts, however, new ideas arise, and the goal of assuring the preservation of female reproductive potential appears a more realistic possibility. This review surveys and discusses advances and challenges of these technologies that, starting from a simple attempt, are now approaching the biosynthesis of a functional engineered ovary.

Author(s): Conca Dioguardi, Carola; Uslu, Bahar; Haynes, Monique; Kurus, Meltem; Gul, Mehmet; Miao, De-Qiang; De Santis, Lucia; Ferrari, Maurizio; Bellone, Stefania; Santin, Alessandro; Giulivi, Cecilia; Hoffman, Gloria; Usdin, Karen; Johnson, Joshua | Abstract: Study hypothesisWe hypothesized that the mitochondria of granulosa cells (GC) and/or oocytes might be abnormal in a mouse model of fragile X premutation (FXPM).Study findingMice heterozygous and homozygous for the FXPM have increased death (atresia) of large ovarian follicles, fewer corpora lutea with a gene dosage effect manifesting in decreased litter size(s). Furthermore, granulosa cells (GC) and oocytes of FXPM mice have decreased mitochondrial content, structurally abnormal mitochondria, and reduced expression of critical mitochondrial genes. Because this mouse allele produces the mutant Fragile X mental retardation 1 (Fmr1) transcript and reduced levels of wild-type (WT) Fmr1 protein (FMRP), but does not produce a Repeat Associated Non-ATG Translation (RAN)-translation product, our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary.What is known alreadyMitochondrial dysfunction has been detected in somatic cells of human and mouse FX PM carriers and mitochondria are essential for oogenesis and ovarian follicle development, FX-associated primary ovarian insufficiency (FXPOI) is seen in women with FXPM alleles. These alleles have 55-200 CGG repeats in the 5' UTR of an X-linked gene known as FMR1. The molecular basis of the pathology seen in this disorder is unclear but is thought to involve either some deleterious consequence of overexpression of RNA with long CGG-repeat tracts or of the generation of a repeat-associated non-AUG translation (RAN translation) product that is toxic.Study design, samples/materials, methodsAnalysis of ovarian function in a knock-in FXPM mouse model carrying 130 CGG repeats was performed as follows on WT, PM/+, and PM/PM genotypes. Histomorphometric assessment of follicle and corpora lutea numbers in ovaries from 8-month-old mice was executed, along with litter size analysis. Mitochondrial DNA copy number was quantified in oocytes and GC using quantitative PCR, and cumulus granulosa mitochondrial content was measured by flow cytometric analysis after staining of cells with Mitotracker dye. Transmission electron micrographs were prepared of GC within small growing follicles and mitochondrial architecture was compared. Quantitative RT-PCR analysis of key genes involved in mitochondrial structure and recycling was performed.Main results and the role of chanceA defect was found in follicle survival at the large antral stage in PM/+ and PM/PM mice. Litter size was significantly decreased in PM/PM mice, and corpora lutea were significantly reduced in mice of both mutant genotypes. Mitochondrial DNA copy number was significantly decreased in GC and metaphase II eggs in mutants. Flow cytometric analysis revealed that PM/+ and PM/PM animals lack the cumulus GC that harbor the greatest mitochondrial content as found in wild-type animals. Electron microscopic evaluation of GC of small growing follicles revealed mitochondrial structural abnormalities, including disorganized and vacuolar cristae. Finally, aberrant mitochondrial gene expression was detected. Mitofusin 2 (Mfn2) and Optic atrophy 1 (Opa1), genes involved in mitochondrial fusion and structure, respectively, were significantly decreased in whole ovaries of both mutant genotypes. Mitochondrial fission factor 1 (Mff1) was significantly decreased in PM/+ and PM/PM GC and eggs compared with wild-type controls.Limitations, reasons for cautionData from the mouse model used for these studies should be viewed with some caution when considering parallels to the human FXPOI condition.Wider implications of the findingsOur data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary. FXPM disease states, including FXPOI, may share mitochondrial dysfunction as a common underlying mechanism.Large scale dataNot applicable.Study funding and competing interestsStudies were supported by NIH R21 071873 (J.J./G.H), The Albert McKern Fund for Perinatal Research (J.J.), NIH Intramural Funds (K.U.), and a TUBITAK Research Fellowship Award (B.U.). No conflict(s) of interest or competing interest(s) are noted.

We describe successful controlled ovarian stimulation (COS) and the first known IVF pregnancy in a trisomy X carrier with associated hypogonadotropic hypogonadism (HH) linked to a chromosome 4 double mutation in the allele of the Gonadotropins Releasing Hormone receptor (GnRHr) gene. Previous administration of low dose of gonadotropins, as recommended in patients with HH, led to poor follicular recruitment. Since trisomy X is a risk factor for diminished ovarian reserve (DOR) and premature ovarian insufficiency (POI), higher doses of gonadotropins led to better ovarian response. The report readknowledges the importance of a correct genetic evaluation in a competent laboratory as a reliable base for treatment planning in this kind of patients.

Carbon monoxide acute intoxication is a common cause of accidental poisoning in industrialized countries and sometimes it produces a real mass casualty incident. The incident described here occurred in a church in the province of Verona, when a group of people was exposed to carbon monoxide due to a heating system malfunction. Fifty-seven people went to the Emergency Department. The mean carboxyhemoglobin (COHb) level was 10.1±5.7% (range: 3-25%). The clinicians, after medical examination, decided to move 37 patients to hyperbaric chambers for hyperbaric oxygen (HBO) therapy. This is the first case report that highlights and analyses the logistic difficulties of managing a mass carbon monoxide poisoning in different health care settings, with a high influx of patients in an Emergency Department and a complex liaison between emergency services. This article shows how it is possible to manage a complex situation with good outcome. (Disaster Med Public Health Preparedness. 2017;11:251–255)

We report on a 31-year old female who presented at genetic counseling for a small uterus, secondary amenorrhea and sterility. Gonadotropic hormone levels were low, suggesting a Hypogonadotropic Hypogonadism (HH) condition. Cytogenetic analysis demonstrated the presence of Trisomy X associated to an interstitial deletion of chromosome 4q13.2, resulting in the complete loss of a copy of the GNRHR gene. As GNRHR is known to be responsible for an autosomal recessive form of HH, we checked the status of the undeleted allele and we found the Q106R substitution. In conclusion, the results of our cytogenetic and molecular analyses have allowed us to clarify the etiology of the patient's condition.

In human IVF, cryopreservation of oocytes has become an alternative to embryo storage. It has also shown enormous potential for oocyte donation, fertility preservation and animal biotechnology. Mouse oocytes have represented the elective model to develop oocyte cryopreservation in the human and over several decades their use has made possible the development of theoretical and empirical approaches. Progress in vitrification has overshadowed slow freezing to such an extent that it has been suggested that vitrification could soon become the exclusive cryopreservation choice in human IVF. However, recent studies have clearly indicated that human embryo slow freezing, a practice considered well established for decades, can be significantly improved by a simple empirical approach. Alternatively, recent and more advanced theoretical models can predict oocyte responses to the diverse factors characterizing an entire slow-freezing procedure, offering a global method for the improvement of current protocols. This gives credit to the notion that oocyte slow freezing still has considerable margins for improvement. In human IVF, cryopreservation of oocytes has become an alternative to embryo storage. It has also shown enormous potential for oocyte donation, fertility preservation and animal biotechnology. Mouse oocytes have represented the elective model to develop oocyte cryopreservation in the human and over several decades their use has made possible the development of theoretical and empirical approaches. Progress in vitrification has overshadowed slow freezing to such an extent that it has been suggested that vitrification could soon become the exclusive cryopreservation choice in human IVF. However, recent studies have clearly indicated that human embryo slow freezing, a practice considered well established for decades, can be significantly improved by a simple empirical approach. Alternatively, recent and more advanced theoretical models can predict oocyte responses to the diverse factors characterizing an entire slow freezing procedure, offering a global method for the improvement of current protocols. This gives credit to the notion that oocyte slow freezing still has considerable margins of improvement.

Objective To evaluate the efficacy of oocyte cryopreservation by a single slow-cooling protocol involving sucrose (0.2 mol/L) in the freezing solution. Design Observational comparison of the clinical outcome in fresh and frozen thawed cycles. Setting Public and private IVF centers. Patient(s) Infertile couples undergoing IVF treatment. Intervention(s) Use of a maximum three oocytes in fresh cycles, as established by local law, and cryopreservation and later use of surplus oocytes. Likewise fresh cycles, maximum three thawed oocytes were used per cycle. All thawed oocytes were microinjected. Main Outcome Measure(s) Embryologic and clinical parameters of fresh and thawed cycles. Result(s) Two thousand forty-six patients underwent 2,209 oocyte retrievals involving oocyte cryopreservation. Overall, the survival rate of thawed oocytes was 55.8%. In 940 thaw cycles, the mean numbers of inseminated oocytes and fertilization rates were significantly decreased vs. fresh cycles outcomes (2.6 ± 0.7 vs. 2.9 ± 0.2 and 72.5% vs. 78.3%, respectively), as were the rates of implantation (10.1% vs. 15.4%), pregnancy rates per transfer (17.0% vs. 27.9%), and pregnancy rates per cycle (13.7% vs. 26.2%). Differences in clinical outcome were found among centers. A pregnancy rate per thawing cycle above 14% was achieved by most clinics. Fifty-seven retrievals involving oocyte cryopreservation achieved a pregnancy after fresh embryo replacement. Implantation and pregnancy rates per embryo transfer and per thawing cycles were 17.5%, 28.6%, and 24.6%, respectively. Conclusion(s) Under the conditions tested, the clinical outcome of oocyte slow-cooling cryopreservation is reduced compared with fresh cycles. Nevertheless, in cases of inapplicability of embryo cryopreservation, oocyte cryopreservation should be offered to patients with surplus oocytes.

Polycystic ovary syndrome (PCOS) is often characterized by chronic oligo- or anovulation (usually manifested as oligo- or amenorrhea), and hyperandrogenism. In addition, 30-40% of PCOS women have impaired glucose tolerance, and a defect in the insulin signaling pathway (inositol-containing phosphoglycan mediators) seems to be implicated in the pathogenesis of insulin resistance. PCOS patients are subfertile as a consequence of such ovulatory disorders and often need drugs, such as clomiphene citrate or follicle-stimulating hormone, for ovulation induction, which increases the risk of multiple pregnancy and ovarian hyperstimulation syndrome. We hypothesized that the administration of an isoform of inositol (myo-inositol), belonging to the vitamin B complex, would improve the insulin-receptor activity, restoring normal ovulatory function.Twenty-five PCOS women of childbearing age with oligo- or amenorrhea were enrolled in the study. Ovulatory disorder due to PCOS was apparently the only cause of infertility; no tubal defect or deficiency of male semen parameters was found. Myo-inositol combined with folic acid (Inofolic) 2 g twice a day was administered continuously. During an observation period of 6 months, ovulatory activity was monitored with ultrasound scan and hormonal profile, and the numbers of spontaneous menstrual cycles and eventually pregnancies were assessed.Twenty-two out of the 25 (88%) patients restored at least one spontaneous menstrual cycle during treatment, of whom 18 (72%) maintained normal ovulatory activity during the follow-up period. A total of 10 singleton pregnancies (40% of patients) were obtained. Nine clinical pregnancies were assessed with fetal heart beat at ultrasound scan. Two pregnancies evolved in spontaneous abortion.Myo-inositol is a simple and safe treatment that is capable of restoring spontaneous ovarian activity and consequently fertility in most patients with PCOS. This therapy did not cause multiple pregnancy.

Background Rare copy number variations (CNVs) are today recognized as an important cause of various neurodevelopmental disorders, including mental retardation and epilepsy. In some cases, a second CNV may contribute to a more severe clinical presentation. Results Here we describe a patient with epilepsy, mental retardation, developmental disorders, and dysmorphic features, who inherited a deletion of 16p13.11 and a triplication of 19p13.3 from his father and mother, respectively. The mother presented mild mental retardation and language delay too. Conclusions We discuss the phenotypic consequences of the two CNVs and suggest that their synergistic effect is likely responsible for the complicated clinical features observed in our patient.

Women with polycystic ovary syndrome (PCOS) typically have abnormal gonadotropin secretion and, in 30% of cases, mild and transient hyperprolactinemia. Elevated prolactin (PRL) is found in the late follicular and luteal phases of natural as well as stimulated cycles. Increased levels of both PRL and luteinizing hormone (LH) conceivably reflect a reduced dopamine inhibitory effect. To clarify the influence of dopaminergic control in PCOS, the investigators retrospectively examined the effects of cabergoline, a potent dopamine agonist that inhibits PRL secretion, on the ovarian response to stimulation with recombinant follicle-stimulating hormone (FSH). Enrolled in the study were 44 couples in which the women were diagnosed as having PCOS, in part because of anovulatory infertility and/or oligomenorrhea or amenorrhea. Eighteen women (group A) whose mean serum PRL was 32. 8 ng/ml received 0.25 mg of cabergoline (Dostinex) per week before and during ovarian stimulation. The remaining 26 women (group B), with a mean PRL of 31.3 ng/ml, received no treatment for hyperprolactinemia. For ovarian stimulation investigators used Triptorelin, a gonadotropin-releasing hormone agonist; FSH in an individualized dose; and human chorionic gonadotropin (hCG). The two groups were similar demographically and clinically. The total of 107 ovulatory-stimulated cycles available for study represented approximately 2 ½ per participant. Cabergoline reduced the mean serum prolactin to 12.5 ng/ml at the time stimulation began. Women not given cabergoline received significantly less recombinant FSH per cycle and were stimulated for a shorter time before hCG was given. Their peak serum estradiol levels were significantly higher than in group A women. Serum progesterone levels on the day of hCG treatment did not differ significantly in the two groups. Ultrasonography showed that hyperprolactinemic women developed significantly more follicles of all sizes on the day hCG was given than did those given cabergoline. Mild ovarian hyperstimulation syndrome (OHSS) developed in 6.2% of group B women and 2.4% of those in group A; all these cycles were canceled. There were 8 pregnancies in group A, 3 of them multiple, and 11 in group B, 5 of them multiple; the differences were not significant. These findings agree with the existence of a dopaminergic component in the regulation of LH release in women with PCOS. Pretreatment with the dopamine agonist cabergoline seems to reduce the ovarian response to recombinant FSH without altering the pregnancy rate or the risk of multiple pregnancy. This might be a helpful means of limiting the risk of OHSS.

In view of the frequent absence of symptoms related to pancreatic lesions, screening tests for VHL should always include assessment of the pancreas and, considering the frequency of polycystic manifestations, VHL should always be borne in mind in the differential diagnosis of multiple pancreatic cysts, especially when occurring in young patients and in the absence of a positive history of pancreatic disease. Von Hippel-Lindau disease (VHL) is a hereditary disease transmitted with an autosomal dominant character and characterized by hemangioblastomas of the central nervous system and retina, renal tumors and cysts, and pheochromocytoma. Pancreatic manifestations of VHL are reported in the literature with incidences ranging from 16 to 29% of cases and consist mainly in cystadenomas of the serous type and in multiple cystic lesions, often with complete replacement of the gland. We report five cases of VHL with a polycystic pancreas as the main or only manifestation, all devoid of symptoms related to involvement of the pancreas, who were referred to our Pancreatic Surgery center with diagnoses of multiple pancreatic pseudocysts of undefined origin.

Background. The aim of this study was to evaluate hysteroscopy routinely performed prior to in vitro fertilization-embryo transfer (IVF-ET).Methods. We analyzed in a prospective study 300 patients who underwent hysteroscopy before the first IVF-ET cycle. We analyzed then in a retrospective manner 300 patients who did not perform hysteroscopy.Results. One-hundred-and-eighty (60%) hysteroscopies were normal but 120 (40%) revealed an unsuspected intrauterine abnormality. We did not find statistically significant differences between patients with normal or abnormal hysteroscopy in any characteristic. We found a statistically significant difference in pregnancy rate between women who performed hysteroscopy before IVF-ET cycle and in women who did not perform it.Conclusions. Hysteroscopy, as a routine examination, should be performed before the first IVF-ET cycle in all patients.

Mammalian oocytes acquire a series of competencies during follicular development that play critical roles at fertilization and subsequent stages of preimplantation embryonic development. Before the mammalian oocyte engages in the fertilization process, it must acquire an array of molecular and cellular assets defining its developmental potential. These properties specify competencies to complete meiosis and initiate mitosis. Meiotic maturation requires the acquisition both of nuclear and cytoplasmic competence and this complex mechanism involves most of the organelles, the cytoskeleton and molecules that are relocated from the nucleus to the cytoplasm. Messenger RNAs of maternal origin are accumulated in the oocyte throughout its growth in the ovary. These transcripts are then shuttled to specific sites of the ooplasm, where local translation is promoted. The nucleus as an organelle undergoes significant positional rearrangements during maturation. At the same time, the spindle changes its localization drifting from the cortical region to the centre of the oocyte and then back cortically. The endoplasmic reticulum undergoes significant changes during maturation in its distribution that reflect an enhanced ability to release Ca2+. Energy derives from cellular ATP as a result of mitochondrial activity. Mitochondria are themselves subject to redistribution and changes in activity during transition from the germinal vesicle to the metaphase II stage, in a fashion that can profoundly influence the quality of the mature oocyte and the destiny of the ensuing embryo. The dynamics of Golgi membranes during meiosis is still partially unknown but generally, as it occurs in the bovine, involves movements from the centre of the cytoplasm to the cortical area. Afterwards, a second translocation occurs between germinal vesicle breakdown and metaphase I stages, repositioning the Golgi from the periphery to the central cytoplasmic area.

Assisted reproduction technology (ART) is becoming an increasingly important strategy to preserve female fertility. Until a decade ago, in an ART context, the only option available to preserve fertility for a woman at risk of loss of ovarian function was embryo cryopreservation. The storage of embryos is a sound, well established, and safe procedure, but it entails major downsides. More recently, new avenues have been opened to preserve female fertility through ART. Ovarian tissue cryopreservation and orthotopic re-implantation have shown the potential to restore ovarian function for a period of time and offer a chance for natural conception. However, so far, only very few full-term pregnancies have been achieved. Therefore, the efficacy of this approach remains to be established. Oocyte cryopreservation is powerfully emerging as an efficient ART procedure able to preserve female fertility without the implications which afflict embryo cryopreservation. Historically, control rate slow cooling has been the technological standard of human embryo cryopreservation. This has marked the early history of oocyte cryopreservation. Consistent advances of control rate slow-cooling protocols have given oocyte cryopreservation the dignity of a routine procedure in many human IVF laboratories. Progress in the last few years has been also driven by the development of the vitrification approach.

In human IVF, cryopreservation of oocytes has become an alternative to embryo storage. It has also shown enormous potential for oocyte donation, fertility preservation and animal biotechnology. Mouse oocytes have represented the elective model to develop oocyte cryopreservation in the human and over several decades their use has made possible the development of theoretical and empirical approaches. Progress in vitrification has overshadowed slow freezing to such an extent that it has been suggested that vitrification could soon become the exclusive cryopreservation choice in human IVF. However, recent studies have clearly indicated that human embryo slow freezing, a practice considered well established for decades, can be significantly improved by a simple empirical approach. Alternatively, recent and more advanced theoretical models can predict oocyte responses to the diverse factors characterizing an entire slow-freezing procedure, offering a global method for the improvement of current protocols. This gives credit to the notion that oocyte slow freezing still has considerable margins for improvement. In human IVF, cryopreservation of oocytes has become an alternative to embryo storage. It has also shown enormous potential for oocyte donation, fertility preservation and animal biotechnology. Mouse oocytes have represented the elective model to develop oocyte cryopreservation in the human and over several decades their use has made possible the development of theoretical and empirical approaches. Progress in vitrification has overshadowed slow freezing to such an extent that it has been suggested that vitrification could soon become the exclusive cryopreservation choice in human IVF. However, recent studies have clearly indicated that human embryo slow freezing, a practice considered well established for decades, can be significantly improved by a simple empirical approach. Alternatively, recent and more advanced theoretical models can predict oocyte responses to the diverse factors characterizing an entire slow freezing procedure, offering a global method for the improvement of current protocols. This gives credit to the notion that oocyte slow freezing still has considerable margins of improvement.

Objective: To determine the effects of myo-inositol on oocyte quality in polycystic ovary syndrome (PCOS) patients undergoing intracytoplasmic sperm injection (ICSI) cycles. Design: A prospective, controlled, randomized trial. Setting: Assisted reproduction centers. Patient(s): Sixty infertile PCO patients undergoing ovulation induction for ICSI. Intervention(s): All participants underwent standard long protocol. Starting on the day of GnRH administration, 30 participants received myo-inositol combined with folic acid (Inofolic) 2 g twice a day and 30 control women received folic acid alone, administrated continuously. Main Outcome Measure(s): Primary end points were number of morphologically mature oocytes retrieved, embryo quality, and pregnancy and implantation rates. Secondary end points were total number of days of FSH stimulation, total dose of gonadotropin administered, E2 level on the day of hCG administration, fertilization rate per number of retrieved oocytes, embryo cleavage rate, live birth and miscarriage rates, cancellation rate, and incidence of moderate or severe ovarian hyperstimulation syndrome. Result(s): Total r-FSH units (1,958 � 695 vs. 2,383 � 578) and number of days of stimulation (11.4 � 0.9 vs. 12.4 � 1.4) were significantly reduced in the myo-inositol group. Furthermore, peak E2 levels (2,232 � 510 vs. 2,713 � 595pg/mL)athCGadministrationweresignificantlylowerinpatientsreceivingmyo-inositol.Themeannumberof oocytes retrieved did not differ in the two groups, whereas in the group cotreated with myo-inositol the mean number of germinal vesicles and degenerated oocytes was significantly reduced (1.0 � 0.9 vs. 1.6 � 1.0), with a trend for increased percentage of oocytes in metaphase II (0.82 � 0.11% vs. 0.75 � 0.15%). Conclusion(s): These data show that in patients with PCOS, treatment with myo-inositol and folic acid, but not folic acid alone, reduces germinal vesicles and degenerated oocytes at ovum pick-up without compromising total number of retrieved oocytes. This approach, reducing E2 levels at hGC administration, could be adopted to decrease therisk of hyperstimulationin such patients. (Fertil Steril� 2009;91:1750‐4. � 2009byAmerican Society for Reproductive Medicine.)

Poor ovarian response to standard in vitro fertilization-embryo transfer (IVF-ET) protocols or different regimens of treatment, as consequence of a diminished ovarian reserve, correlates strictly with patient age, elevated follicle-stimulating hormone (FSH) and reduced antral follicle count. The aim of the present pilot study was to evaluate the outcome of patients with poor prognostic features undergoing IVF-ET with natural cycles as a first approach and not as a consequence of a previous failure treatment.Eighteen aged patients (mean +/- standard deviation 40.2 +/- 0.7 years, range 37-43 years) with elevated serum FSH and reduced antral follicle count underwent intracytoplasmic sperm injection (ICSI) after spontaneous ovulation.A total of 26 natural cycles with ICSI were analyzed. Pregnancy was observed in three patients, of which two were ongoing as assessed by fetal heart beat at ultrasound scan performed 4-5 weeks after ET.The overall pregnancy rates achieved (11.5% per cycle, 20.0% per ET) are comparable with those of conventional IVF-ET in aged patients, and not impaired by a single embryo transferred. Better embryo quality, as a consequence of natural selection of oocytes, better endometrium receptivity and monthly repeatability of the procedure, can balance the relatively low chance to perform ET.

It has been suggested that first polar body (PBI) morphology reflects oocyte competence. Oocytes with an intact normal-sized PBI have been described as generating better day 2 embryos, higher blastocyst yield, and increased pregnancy and implantation rates. In other studies, PBI morphology was found to be unrelated to fertilization rate, embryo quality, and blastocyst formation. In a prospective analysis, the predictive value of the PBI was investigated by comparing the development of oocytes retrieved from intracytoplasmic sperm injection patients and displaying different PBI morphology, classified according to the following characteristics: normal size and smooth surface (I), fragmented (II), rough surface (III), or large size (IV). Fertilization rates were 59, 57, 64 and 60% respectively. No significant differences were found between the various groups. The proportions of high quality (grade A) day 2 embryos were also comparable among groups I-III (14, 12 and 17% respectively), while the low number of grade A embryos in group IV (two embryos) did not allow comparison with the other classes. These data do not suggest that PBI selection can contribute to identification of embryos with high developmental ability. In order to establish alternative criteria for oocyte selection, a metaphase II (MII) spindle analysis was also conducted via Polscope. In oocytes of patients of different age, spindle retardance (which reflects the high order and density of microtubules) was compared with parameters of embryo development. In aged patients, a trend was observed between low retardance and poor embryo quality, although in general the association between retardance and oocyte developmental performance did not reach statistical significance.

The decline of female fertility with advancing age is well documented. The aim of this study was to compare the ovarian performance after repeated ovarian stimulation cycles in women of different ages.Four hundred patients who started at least three in vitro fertilization (IVF) cycles during the 5-year period between 1998 and 2002 were identified. The patients were divided into four groups: the 25-30 age group (n = 90), the 31-35 age group (n = 150), the 36-40 age group (n = 110) and the 41-45 age group (n = 50).Comparing subsequent cycles versus the first treatment cycle we found a statistically significantly increased number of ampules of recombinant follicle stimulating hormone (rFSH) needed to reach follicles maturation (p0.001). The number of ampules of gonadotropin required was significantly higher (p0.001) in the groups of advanced age compared with the groups of young women. For women in the 36-40 group and in the 41-45 group we found the number of follicles, the number of oocytes and the proportion of grade A embryos, in every cycle, were significantly lower than in the groups of young women. We compared the characteristics of ovarian stimulation and response of a single age group in different consecutive cycles. We found significant differences (p0.05) only in the number of ampules required.Maternal age adversely affected ovarian performance. During repeated IVF cycles we also noted an age-independent decline of ovarian response.