Algorithm-based individualization methodology of the starting gonadotropin dose in IVF/ICSI and the freeze-all strategy prevent OHSS equally in normal responders: a systematic review and network meta-analysis of the evidence.

PURPOSE: Ovarian hyperstimulation syndrome (OHSS) represents a rare but dangerous condition associated with controlled ovarian stimulation (COS) in IVF/ICSI. Over the last decades, many strategies have been introduced into clinical practice with the objective of preventing this potentially life-threatening condition. Among these, the freeze-all policy has gained great popularity, thanks to improvements in vitrification. Nevertheless, not all clinics have adequate skills in vitrification procedures and patients may be dissatisfied with a longer time to pregnancy. METHODS: This study is a systematic review and network meta-analysis of randomized controlled trials comparing different strategies of ovarian stimulation in IVF/ICSI cycles (freeze-all policy, algorithm-based individualization of the starting dose, experience-based individualization of the starting dose, standard dose) in terms of reduction of OHSS, in normal responders. RESULTS: The results indicate that only the algorithm-based individualization of the starting gonadotropin dose reduces OHSS similarly to the freeze-all strategy. CONCLUSION: Albeit in the era of the freeze-all policy, the personalization of the starting gonadotropin dose obtained by the use of algorithms should be pursued as a valid and safe option for IVF.

The Gender Gap in the Diagnostic-Therapeutic Journey of the Infertile Couple.

Medical procreation impairs both the biological and psychological lives of couples. However, male and female attitudes to infertility are different and require a different approach during the IVF journey. Thus, the gender impact assessment (GIA) method was used to analyse original studies present in the literature. We found some gender-related differences and, subsequently, possible outcomes of intervention to improve healthy reproduction management and prevent infertility. In particular, it became apparent that there was the need for an in-depth male infertility assessment and a gender-specific follow-up.

How fixed versus variable gonadotropin dose during controlled ovarian stimulation could influence the management of infertility patients undergoing IVF treatment: a national Delphi consensus.

AIM: Define how and when fixed starting gonadotropin doses can be used in current clinical ART practices in Italy. METHODS: A Delphi conference consisting of three rounds was performed in order to define the ideal clinical conditions in which fixed-gonadotropin-dose during COS should be applied. During the conference, 19 statements about the current ART practice were provided to a panel of twenty-nine national experts. Median score was 5 (IQ:4-6) in all Delphi rounds. RESULTS: Eleven statements (57.9%) were classified as shareable with high-degree of convergence, 2 (10.5%) as shareable with low convergence and 6 (31.6%) as un-shareable with high convergence. The panel reached high consensus regarding some statements: (i) fixed FSH-dose in normoresponders and poor-responder, (ii) importance of predicting ovarian response before COS, considering multiple markers to select the right stimulation protocol for each patient, (iii) importance of therapy simplification and standardization to improve efficiency during COS. Moreover, a low-convergence was reached about use of GnRH antagonist as first treatment line and drug storage at room temperature. However from these findings, the debate remains open regarding some other statements: (a) usefulness of Bologna-criteria to define poor-responders; (b) efficacy to change always stimulation protocol after a failure IVF; (c) utility of AMH-dosed with standardized automatic mode to define normo-responder patients; (d) usefulness to modify the dosage of 12.5 IU/die during COS to improve stimulation effectiveness. CONCLUSION: Controlled ovarian stimulation remains a challenging clinical step in Assisted Reproductive Technique, especially in some specific patient groups for which no clinical consensus is available. This study is the first attempt to describe the shared clinical opinion regarding the fixed versus variable gonadotropin dose in the real IVF practice.

From telescope to binoculars. Dyadic outcome resulting from psychological counselling for infertile couples undergoing ART.

OBJECTIVE: This longitudinal study aims to evaluate the effect of psychological counselling on quality of life, marital satisfaction and need for parenthood in couples undergoing fertility treatments (ART). BACKGROUND: Recent guidelines on the ART suggest that psychological counselling should target both members of the infertile couple in order to improve their conjoint management of the infertility-related stress. However, studies on the dyadic outcome of couples are scarce. METHODS: 262 patients were originally considered in the study and completed questionnaires on quality of life, need for parenthood and marital satisfaction, before treatment (T1) and at the day of intrauterine insemination/embryo transfer (T2). For the purposes of this study, 34 counselled couples were then matched to 34 non-counselled couples by propensity scores. The Common Fate Model (CFM) was used to examine dyadic changes. RESULTS: Couples receiving counselling had higher dyadic quality of life and lower dyadic stress due to the need for parenthood at T2 compared to non-counselled couples. No differences were found on marital satisfaction. CONCLUSION: The findings provide support for the effectiveness of counselling on interpersonal outcome. The CFM allows researchers to examine how the dyad as a whole responds to counselling, highlighting the change in the couple's relational dynamics.

A randomized controlled trial investigating the use of a predictive nomogram for the selection of the FSH starting dose in IVF/ICSI cycles.

The number of oocytes retrieved is a relevant intermediate outcome in women undergoing IVF/intracytoplasmic sperm injection (ICSI). This trial compared the efficiency of the selection of the FSH starting dose according to a nomogram based on multiple biomarkers (age, day 3 FSH, anti-Müllerian hormone) versus an age-based strategy. The primary outcome measure was the proportion of women with an optimal number of retrieved oocytes defined as 8-14. At their first IVF/ICSI cycle, 191 patients underwent a long gonadotrophin-releasing hormone agonist protocol and were randomized to receive a starting dose of recombinant (human) FSH, based on their age (150 IU if ≤35 years, 225 IU if >35 years) or based on the nomogram. Optimal response was observed in 58/92 patients (63%) in the nomogram group and in 42/99 (42%) in the control group (+21%, 95% CI = 0.07 to 0.35, P = 0.0037). No significant differences were found in the clinical pregnancy rate or the number of embryos cryopreserved per patient. The study showed that the FSH starting dose selected according to ovarian reserve is associated with an increase in the proportion of patients with an optimal response: large trials are recommended to investigate any possible effect on the live-birth rate.

The carriers of the A/G-G/G allelic combination of the c.2039 A>G and c.-29 G>A FSH receptor polymorphisms retrieve the highest number of oocytes in IVF/ICSI cycles.

PURPOSE: The objective of this study was the elucidation of the possible role of the single-nucleotide polymorphisms (SNP) at position -29 and 2039 of the FSH receptor gene (FSHR) as independent predictive markers of ovarian response. Indeed, the tailoring of reproductive treatments is crucial for both maximizing the success of IVF patients and obtaining a reduction in hypo- or hyper-response rates. METHODS: This prospective, observational study analyzed the association of -29 and 2039 FSHR polymorphisms with the number of retrieved oocytes in 140 patients attending an IVF/ICSI cycle for severe male factors (≤5,000,000 spermatozoa/mL) or tubal factors at the ANDROS Day Surgery Clinic, Palermo, Italy. RESULTS: The results of this study demonstrate that the genetic combination of A/G for polymorphism c.2039 A>G with G/G for polymorphism c.-29 G>A is significantly associated with the highest number of collected oocytes (p = 0.03). This association was significant even after controlling for the effect of other clinical variables. CONCLUSIONS: The A/G-G/G allelic variant, identified as an independent variable, if confirmed in a larger number of patients, could be considered as a new genetic biomarker, which could increase the efficacy of prediction models for ovarian stimulation.

Identification of embryo-fetal cells in celomic fluid using morphological and short-tandem repeats analysis.

OBJECTIVE: The main problem to wide acceptability of celocentesis as earlier prenatal diagnosis is contamination of the sample by maternal cells. The objective of this study was to investigate the cellular composition of celomic fluid for morphological discrimination between maternal and embryo-fetal cells. METHOD: Celomic fluids were aspired by ultrasound-guided transcervical celocentesis at 7-9 weeks' gestation from singleton pregnancies before surgical termination for psychological reasons. DNA extracted from celomic fluid cells showed the same morphology, and quantitative fluorescent polymerase chain reaction (PCR) assay was performed to evaluate their fetal or maternal origin. RESULTS: Six different types of non-hematological maternal and four different types of embryo-fetal cells were detected. The most common maternal cells were of epithelial origin. The majority of embryo-fetal cells were roundish with a nucleus located in an eccentric position near the wall. These cells were considered to be erythroblasts, probably derived from the yolk sac that serves as the initial site of erythropoiesis. CONCLUSIONS: The combined use of morphology and DNA analysis makes it possible to select and isolate embryo-fetal cells, even when maternal contamination is high. This development provides the opportunity for the use of celocentesis for early prenatal diagnosis of genetic diseases and application of array comparative genomic hybridization. © 2016 John Wiley & Sons, Ltd.

The pellet swim-up is the best technique for sperm preparation during in vitro fertilization procedures.

PURPOSE: The aim of this study was to investigate the most suitable sperm preparation technique to apply in order to obtain a spermatozoon population with minimal DNA damage during in vitro fertilization procedures. We compared four preparation techniques: direct swim-up (DSU), pellet swim-up (PSU), density gradient (DG), and density gradient followed by swim-up (DG-SU), evaluating the effects of each technique on the DNA damage rate, evaluated by DNA fragmentation index of the spermatozoa obtained. METHODS: In this observational study, 98 semen samples from couples undergoing IVF/ICSI cycles were included. Data were collected between April and November 2014 at the ANDROS Day Surgery Clinic, Palermo, Italy. RESULT(S): The percentages of DNA fragmentation were 18.30 ± 10.8 in raw samples, 6.6 ± 5.7 after DSU, 4.2 ± 3.8 after PSU, 12.9 ± 9.9 after DG, and 3.7 ± 4.0 after DG-SU respectively. Compared to the raw evaluation, all the preparation techniques significantly decreased the total rate of the DNA fragmentation (DSU Z = -8.60, P < 0.008; PSU Z = -8.54, P < 0.008; DG Z = -6.42, P < 0.008, and DG-SU Z = -8.60, P < 0.008, respectively). Comparing them, spermatozoa with intact DNA after PSU and DG-SU were significantly higher than after DSU (Z = -7.12, P < 0.008; Z = -6.59, P < 0.008, respectively) and after DG (Z = -8.41, P < 0.008; Z = -8.60, P < 0.008, respectively). The difference between PSU and DG-SU was not significant (Z = -2.21, P = 0.03). CONCLUSION(S): There are, above all, two techniques of sperm preparation which allow for the recovery of spermatozoa with the lowest DNA fragmentation rate. Furthermore, given low costs and reduced time, we believe that PSU is the best option in the treatment of semen samples during IVF/ICSI.

Infertility-related stress, anxiety and ovarian stimulation: can couples be reassured about the effects of psychological factors on biological responses to assisted reproductive technology?

The aim of this prospective, longitudinal study was to examine the association between couples' pre-treatment psychological characteristics (state anxiety and infertility-related stress levels of both partners) and ovarian response during assisted reproductive technology treatment in a well-controlled sample. A total of 217 heterosexual couples (434 patients), suffering from primary infertility and undergoing their first assisted reproductive technology treatment at the Reproductive Medicine Unit of ANDROS Day Surgery Clinic in Palermo (Italy), were recruited. Psychological variables were assessed using the State Scale of State-Trait Anxiety Inventory (STAI-S) and the Fertility Problem Inventory (FPI). The number of follicles ≥ 16 mm in diameter, evaluated by transvaginal ultrasound scan on the eleventh day of the workup, was chosen as the outcome measure. No association between women's level of anxiety and infertility-related stress, and the number of follicles ≥ 16 mm in diameter was found. Moreover, the male partner's infertility stress and anxiety did not influence the relationship between the woman's infertility-related stress, anxiety level and ovarian response. Fertility staff should reassure couples that the woman's biological response to ovarian stimulation is not influenced by either partner's level of psychological distress.

Assessing infertility-related stress: the factor structure of the Fertility Problem Inventory in Italian couples undergoing infertility treatment.

The factor structure of the Fertility Problem Inventory (FPI) and its invariance across gender were examined in Italian couples undergoing infertility treatment. About 1000 subjects (both partners of 500 couples) completed two questionnaires prior to commencing infertility treatment at a private Clinic in Palermo, Italy. Confirmatory Factor Analysis demonstrated that the original factor structure of the FPI was partially confirmed. Two correlated factors (Infertility Life Domains and Importance of Parenthood) were obtained via a post hoc Exploratory Factor Analysis. Finally, the invariance of this factor structure across gender was confirmed. The study supported the relevance of two interrelated factors specific to infertility stress which could help clinicians to focus on the core infertility-related stress domains of infertile couples.

The gene expression profile of cumulus cells reveals altered pathways in patients with endometriosis.

PURPOSE: The objective of this experimental study was to compare the global gene expression profile of CC of mature oocytes in 18 patients with severe endometriosis and CC in 18 control patients affected by a severe male factor. METHODS: For each group, the CC were pooled, RNA was extracted and a microarray performed. For validating the microarray, a quantitative real-time PCR was performed in the CC of an independent set of patients with endometriosis (n = 5) and controls (n = 7). RESULTS: 595 differentially expressed genes (320 down-regulated, 275 up-regulated, p < 0.05, fold change ≥1.5) were identified. The most significant changes were observed in genes involved in the chemokine signaling and cell-cell or cell-extracellular matrix adhesion pathways. Several genes of these pathways were down-regulated in endometriosis. Individual RT-PCR assays confirmed the microarray for ten genes. CONCLUSIONS: Several genes involved in the chemokine mediated-signaling pathway and in the functional cross-talk between CC and the oocyte are down-regulated in endometriosis CC. The impairment of these processes could explain the reduction of oocyte competence in endometriosis. This preliminary knowledge could be the starting point for a more detailed elucidation of the relationship between endometriosis and oocyte competence.

A "four-cycle program" improves the estimate of the cumulative pregnancy rate and increases the number of actual pregnancies in IUI treatment: a cohort study.

OBJECTIVE: To demonstrate that reduction of the cumulative dropout rate (CDR) improves the accuracy of the estimate of the cumulative pregnancy rate (CPR) in a set of four intrauterine insemination (IUI) cycles ("four-cycle program") and increases the total number of pregnancies obtained. STUDY DESIGN: Single-centre retrospective observational cohort study of couples who underwent IUI cycles at the Andros Day Surgery Clinic, Palermo, from 1997 to 2011. The main outcome measure was the calculation of the CPR, with life table analysis, firstly by giving the same probability of pregnancy to the dropouts as the patients who continued the treatment (usual method) and secondly by considering this probability null (conservative method). The difference between these two methods was used to verify the accuracy of the estimate. RESULT(S): In the 15 years, 924 couples underwent 2956 cycles carried out consecutively in a set of four cycles. The CDR was 16%. The CPR was 31.4% with the usual method and 29.1% with the conservative method. The difference between the two estimates was not significant, indicating a high reliability of the results and a good accuracy of the calculation. Furthermore, maintenance of a low CDR permits improvement of the CPR, as was demonstrated by considering scenarios with worse dropout rates. CONCLUSION(S): The "four-cycle program" results in a reduction in the CDR, allowing a better estimation of the CPR, and increases the number of actual pregnancies in IUI. The CPR should become the focus for reporting outcome rates in IUI cycles. Reduction of the dropout rate allows us to give the patient a more reliable and accurate estimate of the pregnancy rate.

Towards an ideal source of mesenchymal stem cell isolation for possible therapeutic application in regenerative medicine.

BACKGROUND: The possibility of obtaining mesenchymal stem cells (MSCs) from fetal tissue such as amniotic fluid, chorionic villi and placenta is well-known and a comparison between MSCs originating in different sources such as fetal tissue and those from bone marrow in terms of yield and function is a topical issue. The mesenchymal stem cells isolated from bone marrow are well-characterized. Unfortunately the low quantitative yield during isolation is a major problem. For this reason, other tissue sources for MSCs are of paramount importance. CONCLUSION: In this review, starting from a description of the molecular and cellular biology of MSCs, we describe alternative sources of isolation other than bone marrow. Finally, we describe the potential therapeutic application of these cells.

Gene expression of stem cells at different stages of ontological human development.

OBJECTIVES: To compare multipotent mesenchymal stem cells (MSCs) obtained from chorionic villi (CV), amniotic fluid (AF) and placenta, with regard to their phenotype and gene expression, in order to understand if MSCs derived from different extra-embryonic tissues, at different stages of human ontological development, present distinct stemness characteristics. STUDY DESIGN: MSCs obtained from 30 samples of CV, 30 of AF and 10 placentas (obtained from elective caesarean sections) were compared. MSCs at second confluence cultures were characterized by immunophenotypic analysis with flow cytometry using FACS CANTO II. The expression of the genes Oct-4 (Octamer-binding transcription factor 4, also known as POU5F1), Sox-2 (SRY box-containing factor 2), Nanog, Rex-1 (Zfp-42) and Pax-6 (Paired Box Protein-6), was analyzed. Real-time quantitative PCR was performed by ABI Prism 7700, after RNA isolation and retro-transcription in cDNA. Statistical analysis was performed using non-parametric test Kruskal-Wallis (XLSTAT 2011) and confirmed by REST software, to estimate fold changes between samples. Each gene was defined differentially expressed if p-value was <0.05. RESULTS: Cells from all samples were negative for haematopoietic antigens CD45, CD34, CD117 and CD33 and positive for the typical MSCs antigens CD13, CD73 and CD90. Nevertheless, MSCs from AF and placentas showed different fluorescence intensity, reflecting the heterogeneity of these tissues. The gene expression of OCT-4, SOX-2, NANOG was not significantly different among the three groups. In AF, REX-1 and PAX-6 showed a higher expression in comparison to CV. CONCLUSIONS: MSCs of different extra-embryonic tissues showed no differences in immunophenotype when collected from second confluence cultures. The expression of OCT-4, NANOG and SOX-2 was not significantly different, demonstrating that all fetal sources are suitable for obtaining MSCs. These results open new possibilities for the clinical use of MSCs derived from easily accessible sources, in order to develop new protocols for clinical and experimental research.

Effectiveness of strict anti-contamination criteria for non-invasive foetal sex determination in a clinical setting.

OBJECTIVE: The aim of this study is to prevent false positive results in non-invasive foetal sex determination from pregnant women's plasma following strict anti-contamination criteria. METHODS: cffDNA from 200 pregnant women (mean gestational age of 8.3 ± 0.2 weeks) was analyzed based on genetic forensic anti-contamination procedures and classical non-invasive foetal sex determination techniques. RESULTS: No false positive or false negative was reported. The sensitivity and the specificity reached both a 100% value. CONCLUSIONS: Prevention of contamination following our anti-contamination criteria is a good practice for certain non-invasive sex determination, using cffDNA.

Endometrial expression of selected genes in patients achieving pregnancy spontaneously or after ICSI and patients failing at least two ICSI cycles.

The objective of this study was to identify the endometrial gene expression profile in receptive phase, which could represent a useful prognostic tool for selecting IVF patients. Endometrial expression of 47 selected genes biopsied during the window of implantation in natural cycles was compared between patients who achieved a successful pregnancy spontaneously or after subsequent intracytoplasmic sperm injection (ICSI) cycles and patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed significantly different levels of expression in 19 genes, five implicated in apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence for these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile, which could be used in the future as a prognostic tool for IVF patients. Gene expression analysis technology has opened new important perspectives on the study of the physiological processes of different tissues and organs. Specifically for the endometrium, it would be really interesting to find out an endometrial gene expression profile of receptive phase, which could be used in future as a useful prognostic tool for selecting IVF patients. To achieve this aim, the objective of the present paper was the comparison of endometrial expression in natural cycles of 47 selected genes between the biopsies of patients who achieved a successful pregnancy, either spontaneously or after subsequent ICSI cycles, and those of patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed a significant different expression in 19 genes: five implicated in programmed cell death, known as apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence of these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile which could be used in the future as a prognostic tool for IVF patients.

Are attachment dimensions associated with infertility-related stress in couples undergoing their first IVF treatment? A study on the individual and cross-partner effect.

STUDY QUESTION: Are attachment anxiety and avoidance dimensions in female and male partners in couples seeking infertility treatment associated with her and his infertility-related stress? SUMMARY ANSWER: Attachment dimensions are significantly associated with several aspects of infertility stress in couples undergoing IVF treatment. WHAT IS KNOWN AND WHAT THIS PAPER ADDS: Attachment dimensions of anxiety and avoidance (where highly anxious individuals fear rejection and are preoccupied with maintaining proximity to their partner and highly avoidant individuals are uncomfortable with intimacy and prefer to maintain distance from their partner) may influence the well being of individuals undergoing IVF/ICSI treatment. This study showed that one partner's attachment dimensions had a direct effect on the infertility-related stress of the other partner. DESIGN: Cross-sectional study of consecutive couples before starting their first IVF/ICSI treatment in 2009-2011 at the ANDROS clinic in Palermo, Italy. PARTICIPANTS AND SETTING: Three hundred and fifty-nine couples undergoing fertility treatments were invited to participate in the research. The final sample comprised 316 females and 316 males who filled out the psychological questionnaires (Experiences in Close Relationships; Fertility Problem Inventory; State scale of State-Trait Anxiety Inventory). The participants included patients who had a primary infertility diagnosis and were about to undergo their first IVF or ICSI treatment. DATA ANALYSIS METHOD: Paired t-tests were used to examine gender differences on the study variables (attachment anxiety, attachment avoidance, infertility stress, state anxiety, etc.). Associations between infertility-related stress and the study variables were explored using hierarchical stepwise multivariate linear regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Attachment anxiety and attachment avoidance were significantly associated with global infertility stress in both women (ß = 0.24, P < 0.01 and ß = 0.27, P < 0.01) and men (ß = 0.23, P < 0.01 and ß = 0.37, P < 0.01). Regarding the cross-partner effects, men's infertility stress and relationship concerns were associated with their partners' attachment avoidance (ß = 0.10 P < 0.05 and ß = 0.12, P < 0.05); and the infertility stress of women and the scores for need of parenthood were associated with their partners' attachment anxiety (ß = 0.14 P < 0.05 and ß = 0.16, P < 0.05). BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION: The study data are cross sectional, and specifically focus on associations between adult attachment style and infertility stress. Treating the data from couples as independent observations may be a limitation of the analysis. Potential moderators of such relationships (e.g. coping strategies, stress appraisal) are not included in this study. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by funds provided by Centro Andros S.r.l., Palermo, Italy. The authors declare no financial or commercial conflicts of interest in this study.

Conjoined twins in a trichorionic quadruplet pregnancy after intracytoplasmic sperm injection and quarter laser-assisted zona thinning.

OBJECTIVE: To describe the occurrence and management of conjoined twins (CTs) in a trichorionic quadruplet pregnancy after the transfer of three embryos obtained by intracytoplasmic sperm injection (ICSI) that had undergone assisted zona hatching (AZH) using a noncontact laser. DESIGN: Case report. SETTING: Private and public IVF centers. PATIENT(S): A 38-year-old nulliparous woman. INTERVENTION(S): IVF, diagnosis by two-dimensional, three-dimensional, and four-dimensional ultrasound, selective termination at 12 weeks gestation. MAIN OUTCOME MEASURE(S): Clinical follow-up and delivery. RESULT(S): After diagnosis, counselling, and selective termination, the subsequent prenatal course was uneventful, and two healthy boys were delivered by elective cesarean section at 38 weeks gestation. CONCLUSION(S): Monozygotic twinning (MZT) and CTs could complicate multifetal pregnancies obtained by assisted reproduction techniques. The relationship between CTs, IVF, and AZH should be better studied to fully understand the mechanisms that lead to MZT (with the possible role of AZH) and CTs in MZT, in particular. Three-dimensional and four-dimensional ultrasound improve prenatal assessment of the condition. Selective embryo reduction is a safe procedure for the management of these pregnancies.