A novel machine-learning framework based on early embryo morphokinetics identifies a feature signature associated with blastocyst development.

BACKGROUND: Artificial Intelligence entails the application of computer algorithms to the huge and heterogeneous amount of morphodynamic data produced by Time-Lapse Technology. In this context, Machine Learning (ML) methods were developed in order to assist embryologists with automatized and objective predictive models able to standardize human embryo assessment. In this study, we aimed at developing a novel ML-based strategy to identify relevant patterns associated with the prediction of blastocyst development stage on day 5. METHODS: We retrospectively analysed the morphokinetics of 575 embryos obtained from 80 women who underwent IVF at our Unit. Embryo morphokinetics was registered using the Geri plus® time-lapse system. Overall, 30 clinical, morphological and morphokinetic variables related to women and embryos were recorded and combined. Some embryos reached the expanded blastocyst stage on day 5 (BL Group, n = 210), some others did not (nBL Group, n = 365). RESULTS: The novel EmbryoMLSelection framework was developed following four-steps: Feature Selection, Rules Extraction, Rules Selection and Rules Evaluation. Six rules composed by a combination of 8 variables were finally selected, and provided a predictive power described by an AUC of 0.84 and an accuracy of 81%. CONCLUSIONS: We provided herein a new feature-signature able to identify with an high performance embryos with the best developmental competence to reach the expanded blastocyst stage on day 5. Clear and clinically relevant cut-offs were identified for each considered variable, providing an objective tool for early embryo developmental assessment.

The effect of extended cryo-storage following vitrification on embryo competence: a systematic review and meta-analysis.

PURPOSE: Few studies explored whether prolonged cryo-storage after vitrification affects embryo competence and perinatal outcomes. This systematic review and meta-analysis aims at highlighting any putative impact of cryo-storage duration on cryo-survival, miscarriage, live birth and major malformations. METHODS: A systematic review was performed using MEDLINE (PubMed), ISI Web of Knowledge, Scopus and Embase databases up to June 2021. Data were combined to obtain a pooled OR, and meta-analysis was conducted using a random effects model. Out of 1,389 screened abstracts, 22 papers were assessed for eligibility, and 5 studies were included (N = 18,047 embryos). Prolonged cryo-storage was defined as > 12 months (N = 3389 embryos). Subgroup analysis was performed for untested vitrified cleavage stage embryos (N = 1739 embryos) and for untested and euploid vitrified blastocysts (N = 13,596 and 2712 embryos, respectively). RESULTS: Survival rate, miscarriage, live birth and major malformation rates were all similar in the two groups. CONCLUSION: These data further support the safety of long-term cryo-storage of human embryos beyond 12 months. This is reassuring for good prognosis patients with surplus embryos, couples seeking a second child from supernumerary embryos and women postponing the transfer for clinical or personal reasons.

Do we trust scientific evidence? A multicentre retrospective analysis of first IVF/ICSI cycles before and after the OPTIMIST trial.

STUDY QUESTION: Has the practice of individualizing the recombinant-FSH starting dose been superseded after the largest randomized controlled trial (RCT) in assisted reproduction technology (ART), the OPTIMIST trial? SUMMARY ANSWER: The OPTIMIST trial has influenced our ART daily practice to a limited degree, but adherence is still generally poor. WHAT IS KNOWN ALREADY: Although the 'one size fits all' approach has been discouraged for decades by most authors, the OPTIMIST study group demonstrated in a large prospective RCT that, in general, dosage individualization does not improve the prospects for live birth, although it may decrease ovarian hyperstimulation syndrome (OHSS) risk in expected high responders. STUDY DESIGN, SIZE, DURATION: Retrospective analysis of all first in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles from 1st January 2017 to 31st December 2018, before and after the OPTIMIST publication on November 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two thousand six hundred and seventy-seven patients, between 18 and 42 years old, undergoing their first IVF-ICSI cycle in seven Italian fertility centres, were included. Patients were allocated to three groups according to their ovarian reserve markers: predicted poor ovarian responders (POR), predicted normo-responders (NR) and expected hyper-responders (HRs). MAIN RESULTS AND THE ROLE OF CHANCE: Between 2017 and 2018, there was an overall increase in prescription of the standard 150 IU dose proposed by the OPTIMIST trial and a reduction in the use of a starting dose >300 IU. After subgroup analysis, the decrease in doses >300 IU remained significant in the POR and NR sub-groups. LIMITATIONS, REASONS FOR CAUTION: The retrospective nature of the study. Physicians need time to adapt to new scientific evidence and a comparison between 2017 and 2019 may have found a greater impact of the Optimist trial, although other changes over the longer time span might have increased confounding. We cannot be sure that the observed changes can be attributed to knowledge of the OPTIMIST trial. WIDER IMPLICATIONS OF THE FINDINGS: Clinicians may be slow to adopt recommendations based on RCTs; more attention should be given to how these are disseminated and promoted. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used for this study. E.P. reports grants and personal fees from MSD, grants from Ferring, from IBSA, grants and personal fees from Merck, grants from TEVA, grants from Gedeon Richter, outside the submitted work. E.S. reports grants from Ferring, grants and personal fees from Merck-Serono, grants and personal fees from Theramex, outside the submitted work. All other authors do not have conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Correction to: Contribution of cryopreservation to the cumulative live birth rate: a large multicentric cycle-based data analysis from the Italian National Registry.

The original article unfortunately contained a mistake. The names of the collaborators were captured as authors of the article.

Contribution of cryopreservation to the cumulative live birth rate: a large multicentric cycle-based data analysis from the Italian National Registry.

PURPOSE: To estimate the contribution of cryopreservation to the cumulative live birth rate (CLBR) after law modification in Italy in the era of vitrification and freeze-all. METHODS: The Italian National Registry performed a cycle-based data collection. Nine Italian IVF clinics were involved incorporating a total of 10,260 fresh cycles performed between January 2015 and April 2016 resulting in 9273 oocyte retrievals and 3266 subsequent warming cycles from the same oocyte retrievals performed up to December 2016. Mean female age was 37 ± 4.3 years. Primary outcome measure was CLBR per oocyte retrieval. Confounding factors were tested in multivariate regression analysis, and the relative impact of cryopreservation to the CLBR in different patient categories was calculated. RESULTS: CLBR per oocyte retrieval was 32.6%, 26.5%, 18.7%, 13.0%, and 5.5% for women younger than 36, aged 36-39, 40-41, and older than 41 years, respectively. The total relative contribution of oocyte/embryo cryopreservation was 40.6% (95% CI 38.41-42.75). An association between maternal age, number of oocytes retrieved, fertilization rate, cryopreservation, and cumulative live birth was shown. When adjusted for confounders, a 2.3-fold increase was observed in the chance of live birth when cryopreservation was performed (OR 2.3; 95% CI 1.99-2.56). In high responder patients (> 15 oocytes retrieved) where freeze-all was applied in 67.6% of cycles to avoid the risk of hyper stimulation syndrome, the relative contribution of vitrification to the CLBR was 80.6%. CONCLUSIONS: Cryopreservation is essential in IVF and should always be available to patients to optimize success rates. Multicentric, cycle-based data analyses are crucial to provide infertile couples, clinicians, and regulatory bodies with accurate information on IVF effectiveness including fresh and cryopreserved cycles.

Patient-centered communication, patient satisfaction, and retention in care in assisted reproductive technology visits.

PURPOSE: To explore the association between patient-centered communication, patients' satisfaction, and retention in care in assisted reproductive technology (ART) visits. METHODS: ART visits at eight Italian clinics were videotaped and coded using the Roter Interaction Analysis System, which includes a Patient-Centered Index (PCI), a summary "patient-centered communication" ratio. After the visit, patients completed a satisfaction questionnaire (SATQ). After 3 months, patients were asked about their retention in care. Spearman correlations and Mann-Whitney tests were used to test associations between the study variables; the open-ended item of SATQ was analyzed through content analysis. RESULTS: Eighty-five visits were videotaped (involving 28 gynecologists and 160 patients). PCI score (µ = 0.51 ± 0.28) revealed a more disease-oriented communication during the visit. Patients reported high levels of satisfaction with the visit and identified in the information provision or in the doctor's humanity or kindness the main reasons of satisfaction. At the follow-up, the majority of the couples declared to have followed the clinicians' recommendations and to have remained related to the ART center. No associations were found among the study variables, except for a lower male satisfaction among couples who declared to have changed ART clinic. CONCLUSIONS: Contrary to what was expected, the style of physician-patient communication was not found to be associated with patient satisfaction and retention in care. However, patients were highly satisfied and engaged. The actual meaning of a communication that is "patient-centered" in the ART context might be wider, including the couples' need for information, as suggested by qualitative findings.

Human recombinant FSH induces chemoresistance in human breast cancer cells via HIF-1α activation†.

Breast cancer patients under 40 years of age who are candidate to chemotherapy with alkylating drugs may undergo controlled ovarian stimulation (COS) with recombinant human follicle-stimulating hormone (rhFSH) in order to get fertility preservation by mature oocyte cryostorage. The direct effect(s) of exogenous rhFSH on the chemosensitivity of breast cancer is currently unknown. To clarify this issue, we incubated four different breast cancer cell lines with rhFSH (10 IU/L, 24 h) and then we exposed them to doxorubicin (DOX) or cyclophosphamide (CPA). The effect(s) of rhFSH on human breast cancer cells treated with DOX or CPA was measured in terms of (1) cell viability, (2) cytotoxicity, (3) multidrug resistance (MDR) genes and proteins expression and activities, and (4) hypoxia-inducible factor 1-alpha (HIF-1α) activation. Pretreatment with rhFSH significantly increased the viability of breast cancer cells after treatment with DOX or CPA, and reduced the lactate dehydrogenase leakage and reactive oxygen species production. Moreover, after preincubation with rhFSH, the MDR proteins (Pgp, MPR1, and BCRP) expression and activity resulted upregulated and the HIF-1α pathway activated. In addition, the use of a widely used HIF-1α inhibitor, the 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), prevented the rhFSH effect on the onset of MDR. Taken together, these observations suggest that a short exposure to rhFSH induces chemoresistance to DOX and CPA in human breast cancer cells via HIF-1α activation.

Resonant inelastic x-ray incarnation of Young's double-slit experiment.

Young's archetypal double-slit experiment forms the basis for modern diffraction techniques: The elastic scattering of waves yields an interference pattern that captures the real-space structure. Here, we report on an inelastic incarnation of Young's experiment and demonstrate that resonant inelastic x-ray scattering (RIXS) measures interference patterns, which reveal the symmetry and character of electronic excited states in the same way as elastic scattering does for the ground state. A prototypical example is provided by the quasi-molecular electronic structure of insulating Ba3CeIr2O9 with structural Ir dimers and strong spin-orbit coupling. The double "slits" in this resonant experiment are the highly localized core levels of the two Ir atoms within a dimer. The clear double-slit-type sinusoidal interference patterns that we observe allow us to characterize the electronic excitations, demonstrating the power of RIXS interferometry to unravel the electronic structure of solids containing, e.g., dimers, trimers, ladders, or other superstructures.

Probabilistic cost-effectiveness analysis of controlled ovarian stimulation with recombinant FSH plus recombinant LH vs. human menopausal gonadotropin for women undergoing IVF.

BACKGROUND: The association of recombinant FSH plus recombinant LH in 2:1 ratio may be used not only to induce ovulation in anovulatory women with hypogonadotropic hypogonadism but also to achieve multiple follicular developments in human IVF. The aim of this analysis was to estimate the cost-effectiveness of Controlled Ovarian Stimulation (COS) with recombinant FSH (rFSH) plus recombinant LH (rLH) in comparison with highly purified human menopausal gonadotropin (HP-hMG) in the woman undergoing in vitro fertilization (IVF) in Italy. METHODS: A probabilistic decision tree was developed to simulate patients undergoing IVF, either using r-FSH + r-LH or HP-hMG to obtain COS. The model considers the National Health System (NHS) perspective and a time horizon equal to two years. Simulations were reported considering the number of retrieved oocytes (5-9, 10-15 and > 15) and transition probabilities were estimated through specific analyses carried out on the population of 848 women enrolled in the real-life. RESULTS: The model estimated that patients undertaking therapeutic protocol with r-FSH + r-LH increase the general success rate (+ 6.6% for pregnancy). The incremental cost-effectiveness ratio (ICER) per quality-adjusted life year (QALY) of r-FSH + r-LH was below the willingness to pay set at €20,000 for all the considered scenarios. CONCLUSIONS: The cost-utility analysis demonstrated that the r-FSH + r-LH is a cost-effective option for the Italian National Health System (NHS).

Doctor-couple communication during assisted reproductive technology visits.

STUDY QUESTION: What are the characteristics of doctor-couple communication content during actual ART visits? SUMMARY ANSWER: Physicians were mainly focused on providing biomedical information, while communication content from couples had a 2-fold focus on providing biomedical information and on positive talk. WHAT IS KNOWN ALREADY: Communication aspects in ART seem crucial for clinical decision-making, retention in care and critical conversations with couples due to low treatment success rates. However, no studies have been carried out on the actual interaction between the doctor and the couple in this context. STUDY DESIGN, SIZE, DURATION: This observational study involved 28 clinicians and 160 patients referred to eight Italian ART clinics during a one-year recruitment period. PARTICIPANTS/MATERIALS, SETTING, METHODS: ART visits at eight Italian clinics were videotaped. The visits were coded using the Roter Interaction Analysis System (RIAS), particularly focusing on RIAS composite categories, verbal dominance and patient-centeredness score. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 85 visits were eligible for analysis (62% acceptance rate), involving 28 clinicians and 160 patients (including 75 couples). The average visit duration was 37 ± 17.7 min. The mean verbal dominance was 1.9 ± 0.86 (range: 0.72-5.74). Physicians mainly focused on providing biomedical information. Communication content from couples had a 2-fold focus on providing biomedical information and on positive talk. The mean of patient centeredness index (PCI) was 0.51 (SD = 0.28; range 0.08-1.77); visits in which the doctor was a woman or the treatment indication was for heterologous fertilization showed higher PCI scores. Overall, females accounted for 67% of all patient talk. Taking this imbalance into account as expected frequencies for each composite category, males reported significantly more utterances in almost all of the socioemotional categories. LIMITATIONS, REASONS FOR CAUTION: These results are preliminary and observational and only regard Italy. Communication during visits may have been biased since the professionals who agreed to participate showed an interest in communication issues. Another limitation is a possible Hawthorne effect due to the fact that participants were aware of being videotaped. WIDER IMPLICATIONS OF THE FINDINGS: Our study showed that ART physicians mainly adopted an informative model of communication and a more disease-oriented approach. Findings revealed the complexity of communication content during ART consultations, given its triadic characteristic in which the third party is also a patient; clinicians should be aware of this complex aspect and of the specific male and female perspectives to be taken into account. The results could be useful for training ART professionals. STUDY FUNDING/COMPETING INTEREST(S): This study was possible thanks to an unconditional grant from Ferring Spa to the Department of Health Sciences, University of Milan. There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Evaluation of equine oocyte developmental competence using polarized light microscopy.

The purpose of this study was to observe in vitro-matured equine oocytes with an objective computerized technique that involves the use of a polarized light microscope (PLM) in addition to the subjective morphological evaluation obtained using a classic light microscope (LM). Equine cumulus-oocyte complexes (COCs, n = 922) were subjected to different in vitro maturation times (24, 36 or 45 h), however, only 36-h matured oocytes were analyzed using CLM. The 36-h matured oocytes that reached maturity were parthenogenetically activated to evaluate the quality and meiotic competence. Average maturation percentages per session in groups 1, 2 and 3 (24-, 36- and 45-h matured oocytes respectively) were 29.31 ± 13.85, 47.01 ± 9.90 and 36.62 ± 5.28%, whereas the average percentages of immature oocytes per session were 28.78 ± 20.17, 7.83 ± 5.51 and 22.36 ± 8.39% respectively. The zona pellucida (ZP) birefringent properties were estimated and correlated with activation outcome. ZP thickness and retardance of the inner layer of the zona pellucida (IL-ZP) were significantly increased in immature oocytes compared with mature oocytes (P < 0.001 and P < 0.01 respectively). The comparison between parthenogenetically activated and non-activated oocytes showed a significant increase in the area and thickness of the IL-ZP in parthenogenetically activated oocytes (P < 0.01). These results show that the 36-h in vitro maturation (IVM) protocol allowed equine oocytes to reach maturity, and PLM observation of ZP can be used to distinguish mature and immature oocytes as well as activated and non-activated oocytes.

Angiogenic properties of endometrial mesenchymal stromal cells in endothelial co-culture: an in vitro model of endometriosis.

Study Question: Can endometrial mesenchymal stromal cells (E-MSCs) differentiate into endothelial cells in an in vitro co-culture system with human umbilical vein endothelial cells (HUVECs)? Summary Answer: E-MSCs can acquire endothelial markers and function in a direct co-culture system with HUVECs. What is Known Already: E-MSCs have been identified in the human endometrium as well as in endometriotic lesions. E-MSCs appear to be involved in formation of the endometrial stromal vascular tissue and the support of tissue growth and vascularization. The use of anti-angiogenic drugs appears as a possible therapeutic strategy against endometriosis. Study Design, Size, Duration: This is an in vitro study comprising patients receiving surgical treatment of ovarian endometriosis (n = 9). Participants/Materials, Setting, Methods: E-MSCs were isolated from eutopic and ectopic endometrial tissue and were characterized for the expression of mesenchymal and endothelial markers by FACS analysis and Real-Time PCR. CD31 acquisition was evaluated by FACS analysis and immunofluorescence after a 48 h-direct co-culture with green fluorescent protein +-HUVECs. A tube-forming assay was set up in order to analyze the functional potential of their interaction. Finally, co-cultures were treated with the anti-angiogenic agent Cabergoline. Main Results and the Role of Chance: A subpopulation of E-MSCs acquired CD31 expression and integrated into tube-like structures when directly in contact with HUVECs, as observed by both FACS analysis and immunofluorescence. The isolation of CD31+ E-MSCs revealed significant increases in CD31, vascular endothelial growth factor receptor 2, TEK receptor tyrosine kinase and vascular endothelial-Cadherin mRNA expression levels with respect to basal and to CD31neg cells (P < 0.05). On the other hand, the expression of mesenchymal genes such as c-Myc, Vimentin, neuronal-Cadherin and sushi domain containing 2 remained unchanged. Cabergoline treatment induced a significant reduction of the E-MSC angiogenic potential (P < 0.05 versus control). Large Scale Data: Not applicable. Limitations, Reasons for Caution: Further studies are necessary to investigate the cellular and molecular mechanisms underlying the endothelial cell differentiation. Wider Implications of the Findings: E-MSCs may undergo endothelial differentiation, and be potentially involved in the development of endometriotic implants. Cell culture systems that more closely mimic the cellular complexity typical of endometriotic tissues in vivo are required to develop novel strategies for treatment. Study Funding/Competing Interest(s): This study was supported by the 'Research Fund ex-60%', University of Turin, Turin, Italy. All authors declare that their participation in the study did not involve actual or potential conflicts of interests.

Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.

STUDY QUESTION: Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? SUMMARY ANSWER: The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. WHAT IS KNOWN ALREADY: ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. STUDY DESIGN, SIZE, DURATION: This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single oocytes and their corresponding CC were analysed. Oocytes were examined using PLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P < 0.05) and ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P < 0.05). This coincided with a significantly smaller IL-ZP area and thickness in mature oocytes than in immature oocytes (all P < 0.05). In mature oocytes, IL-ZP retardance was significantly correlated with the expression of all four ZP mRNAs (all P < 0.05). The oocyte ZP3 expression was the main predictor of the fertilization capacity, next to IL-retardance and IL-thickness. Using stepwise regression analysis, IL-thickness combined with EFNB2 expression in CC and the patient's ovarian response resulted in a noninvasive oocyte fertilization prediction model. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study and the relation of oocyte mRNA levels to fertilization capacity is indirect as oocyte gene expression analysis required lysis of the oocyte. WIDER IMPLICATIONS OF THE FINDINGS: Overall relations between PLM observations, mRNA expression changes and intrinsic oocyte competence were successfully documented. As such PLM and CC gene expression are confirmed as valuable noninvasive techniques to evaluate oocyte competence. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by University of Torino, Italy, WFWG UZ-Brussel and Agentschap voor Innovatie door Wetenschap en Technologie IWT 110680, Belgium. All authors declare that their participation in the study did not involve actual or potential conflicts of interests.

Large randomized trial comparing transabdominal ultrasound-guided embryo transfer with a technique based on uterine length measurement before embryo transfer.

OBJECTIVE: To compare in a randomized, non-inferiority trial the embryo transfer (ET) technique based on uterine length measurement before transfer (ULMbET) with transabdominal ultrasound-guided embryo transfer (TA-UGET) in a large population of patients undergoing in-vitro fertilization (IVF). METHODS: Patients undergoing IVF with ET with cleaving fresh embryos were randomized to receive ULMbET or TA-UGET. The transfer of one to three embryos on day 2-3 of culture was performed using a soft catheter either under transabdominal ultrasound guidance (TA-UGET group) or after measurement of the uterine cavity by transvaginal ultrasound and calculation of the discharge site (ULMbET group). The primary outcome measures were clinical pregnancy rate, ongoing pregnancy rate and implantation rate, and secondary outcomes included patient discomfort during ET. RESULTS: A total of 1648 patients undergoing IVF were randomized to receive ULMbET (n = 828) or TA-UGET (n = 820) and were included in the analysis. Comparable clinical pregnancy rate (38.2% vs 38.9%), implantation rate (24.8% vs 25.2%) and ongoing pregnancy rate (33.1% vs 34.8%) were observed in ULMbET and TA-UGET groups. The discomfort intensity score and the proportion of patients with moderate-to-severe discomfort during ET were significantly higher in the TA-UGET group (2.6 vs 1.5 visual analog scale points and 19.8% vs 1.2%; P = 0.045 and P = 0.003, respectively). CONCLUSION: The ULMbET technique leads to IVF results comparable to those obtained with UGET, but is better tolerated than is TA-UGET and is technically easier to perform for a single operator. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Ovarian tissue cryopreservation in girls undergoing haematopoietic stem cell transplant: experience of a single centre.

Fertility after childhood haemopoietic stem cell transplant (HSCT) is a major concern. Conditioning regimens before HSCT present a high risk (>80%) of ovarian failure. Since 2000, we have proposed cryopreservation of ovarian tissue to female patients undergoing HSCT at our centre, to preserve future fertility. After clinical and haematological evaluation, the patients underwent ovarian tissue collection by laparoscopy. The tissue was analysed by histologic examination to detect any tumour contamination and then frozen following the slow freezing procedure and cryopreserved in liquid nitrogen. From August 2000 to September 2013, 47 patients planned to receive HSCT, underwent ovarian tissue cryopreservation. The median age at diagnosis was 11.1 years and at the time of procedure it was 13 years, respectively. Twenty-four patients were not pubertal at the time of storage, whereas 23 patients had already experienced menarche. The median time between laparoscopy and HSCT was 25 days. Twenty-six out of 28 evaluable patients (93%) developed hypergonadotropic hypogonadism at a median time of 23.3 months after HSCT. One patient required autologous orthotopic transplantation that resulted in one live birth. Results show a very high rate of iatrogenic hypergonadotropic hypogonadism, highlighting the need for fertility preservation in these patients.

Endometrial adult/progenitor stem cells: pathogenetic theory and new antiangiogenic approach for endometriosis therapy.

The cyclical arrival of endometrial cells into the abdominal cavity through retrograde flux at menstruation represents the etiopathogenetic basis of endometriosis. The endometrium has peculiar regenerative properties linked to the presence of adult stem cells similar to mesenchymal stem cells (MSCs). Once in the abdominal cavity, these MSCs could proliferate, invade, and differentiate into endometrial cells, finally generating ectopic implants. As only differentiated endometrial cells, and not endometrial MSCs, possess steroid hormone receptors, MSCs could be responsible for the high rate of persistence/recurrence of the disease after hypoestrogenism-inducing therapies. Even angiogenesis promoted by MSCs could play an important role, as survival and proliferation of endometriotic tissue depend on the formation of new blood vessels. Inhibition of angiogenesis represents, in fact, a new, promising therapeutic approach for the disease. Further, medications directly targeting endometriosis MSCs could be effective, alone or in association with hormonal treatments, in increasing the success of medical treatment.

Is letrozole needed for controlled ovarian stimulation in patients with estrogen receptor-positive breast cancer?

OBJECTIVE: To assess the advantages and disadvantages of using letrozole for controlled ovarian stimulation (COH) in young patients with estrogen receptor-positive (ER+) breast cancer, wishing to cryopreserve oocytes. DESIGN: Retrospective cohort analysis. SETTING: Sixteen Italian units for reproductive medicine and in vitro fertilization. METHODS: Data of 50 ER+ breast cancer patients undergoing COH to cryopreserve oocytes before gonadotoxic chemotherapy with a letrozole plus gonadotropins (Le+Gn) protocol were compared with those of 25 young women with ER- breast cancer, submitted to COH using a protocol with gonadotropins alone (Gn-only). RESULTS: The Le+Gn protocol implied a significantly lower total Gn consumption and allowed to maintain significantly lower circulating E2 levels at all checkpoints throughout stimulation (peak E2 value 446 ± 357 versus 1553 ± 908 pg/ml, respectively; p = 0.001). On the other side, the Le+Gn protocol allowed a significantly lower yield of oocytes available for cryostorage (6.6 ± 3.5 versus 8 ± 5, respectively; p = 0.038). CONCLUSIONS: In breast cancer patients, the association of letrozole to Gn significantly reduces the number of oocytes available for cryostorage in comparison with the use of Gn alone. On the other side, it is associated with significantly lower E2 levels during the whole stimulation cycle, a safety issue that has been traditionally considered advantageous in case of ER+ cancers.

Effect of the method of conception and embryo transfer procedure on mid-gestation placenta and fetal development in an IVF mouse model.

BACKGROUND: Abnormal placentation is a potential mechanism to explain the increased incidence of low birthweight observed after IVF. This study evaluates, in a mouse model, whether the method of conception and embryo transfer affect placentation and fetal development. METHODS: IVF blastocysts (CF1 x B6D2F1/J) were cultured in Whitten's medium (IVF(WM), n = 55) or K modified simplex optimized medium with amino acids (IVF(KAA), n = 56). Embryos were transferred to the uteri of pseudo-pregnant recipients. Two control groups were created: unmanipulated embryos produced by natural mating (in vivo group, n = 64) and embryos produced by natural mating that were flushed from uterus and immediately transferred to pseudo-pregnant recipients (flushed blastocysts, FB group, n = 57). At gestation age 12.5 days, implantation sites were collected and fixed; fetuses and placentas were weighed and their developmental stage (DS) evaluated. Placental areas and vascular volume fractions were calculated; parametric statistics were applied as appropriate. RESULTS: IVF fetuses showed a modest but significant delay in development compared with FB mice (P < 0.05). In addition, IVF conceptuses were consistently smaller than FB (P < 0.05). Importantly, these differences persisted when analyzing fetuses of similar DS. The placenta/fetus ratio was larger in the IVF group (IVF(WM) 0.95; IVF(KAA) = 0.90) than the FB group (0.72) (P < 0.05 for all comparisons). Gross morphology of the placenta and ratio labyrinth/fetal area were equivalent in the IVF and FB groups, as were percentage of fetal blood vessels, maternal blood spaces and trophoblastic components. CONCLUSIONS: In vitro embryo culture affects fetal and placental development; this could explain the lower birthweight in IVF offspring.

Long-term cryostorage does not adversely affect the outcome of oocyte thawing cycles.

This multi-centre study evaluated systematically the influence of the duration of cryostorage on the outcome of thawing cycles when using slow-frozen oocytes. The thawing cycles were retrospectively divided into three main groups based on cryostorage duration: group A, 1-3 months; group B, 4-6 months; and group C, 7-48 months. Group C was subsequently divided into three subgroups: group C1, 7-9 months; group C2, 10-12 months; and group C3, 13-48 months. Main outcome measures observed were oocyte survival after thawing, fertilization, cleavage; embryo quality and development, implantation, and birth. No significant differences in main outcome measures were observed between all the groups studied. In conclusion, human oocytes can be safely cryostored for several years. This finding could encourage the wider use of oocyte cryopreservation as a clinical procedure in assisted reproduction.

Impact of Italian legislation regulating assisted reproduction techniques on ICSI outcomes in severe male factor infertility: a multicentric survey.

BACKGROUND: In 2004, a law regulating assisted reproduction techniques (ART) was passed in Italy. The new rules allow for the formation and transfer of a maximum of three embryos at one time, whereas embryo selection and embryo storage are prohibited. The aim of this study is to evaluate the impact of these restrictions on ICSI outcome in couples affected by severe male factor infertility. METHODS: Thirteen Italian ART Units were involved in this study. Data were collected on ICSI cycles performed during 2 years before (control group) and 2 years after (study group) the enforcement of the law. Only cases of obstructive azoospermia (OA), non-obstructive azoospermia (NOA) and severe oligoastenoteratozoospermia (OAT) (sperm count