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STUDY QUESTION Does LH addition to FSH in vitro recover the human primary granulosa lutein cell (hGLC) sub/poor-response? SUMMARY ANSWER A picomolar concentration of LH may recover the FSH-induced cAMP and progesterone production of hGLC from sub/poor-responder women. WHAT is KNOWN ALREADY Clinical studies suggested that FSH and LH co-treatment may be beneficial for the ovarian response of sub/poor-responders undergoing ovarian stimulation during ART. STUDY DESIGN, SIZE, DURATION hGLC samples from 286 anonymous women undergoing oocyte retrieval for ART were collected from October 2017 to February 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS hGLCs from women undergoing ovarian stimulation during ART were blindly purified, cultured, genotyped and treated in vitro by increasing concentrations of FSH (nM) ±0.5 nM LH. cAMP and progesterone levels produced after 3 and 24 h, respectively, were measured. In vitro data were stratified a posteriori, according to the donors’ ovarian response, into normo-, sub- and poor-responder groups and statistically compared. The effects of LH addition to FSH were compared with those obtained by FSH alone in all the groups as well. MAIN RESULTS AND THE ROLE of CHANCE hGLCs from normo-responders were shown to have higher sensitivity to FSH treatment than sub-/poor-responders in vitro. Equimolar FSH concentrations induced higher cAMP (about 2.5- to 4.2-fold), and progesterone plateau levels (1.2- to 2.1-fold), in cells from normo-responder women than those from sub-/poor-responders (ANOVA; P 0.05). Interestingly, these in vitro endpoints, collected from the normo-responder group treated with FSH alone, were similar to those obtained in the sub-/poor-responder group under FSH + LH treatment. No different allele frequencies and FSH receptor (FSHR) gene expression levels between groups were found, excluding genetics of gonadotropin and their receptors as a factor linked to the normo-, sub- and poor-response. In conclusion, FSH elicits phenotype-specific ovarian lutein cell response. Most importantly, LH addition may fill the gap between cAMP and steroid production patterns between normo- and sub/poor-responders. LIMITATIONS, REASONS FOR CAUTION Although the number of experimental replicates is overall high for an in vitro study, clinical trials are required to demonstrate if the endpoints evaluated herein reflect parameters of successful ART. hGLC retrieved after ovarian stimulation may not fully reproduce the response to hormones of granulosa cells from the antral follicular stage. WIDER IMPLICATIONS of THE FINDINGS This in vitro assay may describe the individual response to personalize ART stimulation protocol, according to the normo-, sub- and poor-responder status. Moreover, this in vitro study supports the need to conduct optimally designed, randomized clinical trials exploring the personalized use of LH in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by Merck KGaA. M.L. and C.C. are employees of Merck KGaA or of the affiliate Merck Serono SpA. Other authors have no competing interests to declare. TRIAL REGISTRATION NUMBER N/A.

Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.

Purpose Several mathematical models have been developed to estimate individualized chances of assisted reproduction techniques (ART) success, although with limited clinical application. Our study aimed to develop a decisional algorithm able to predict pregnancy and live birth rates after controlled ovarian stimulation (COS) phase, helping the physician to decide whether to perform oocytes pick-up continuing the ongoing ART path. Methods A single-center retrospective analysis of real-world data was carried out including all fresh ART cycles performed in 1998–2020. Baseline characteristics, ART parameters and biochemical/clinical pregnancies and live birth rates were collected. A seven-steps systematic approach for model development, combining linear regression analyses and decision trees (DT), was applied for biochemical, clinical pregnancy, and live birth rates. Results Of fresh ART cycles, 12,275 were included. Linear regression analyses highlighted a relationship between number of ovarian follicles > 17 mm detected at ultrasound before pick-up (OF17), embryos number and fertilization rate, and biochemical and clinical pregnancy rates (p

In IVF centers, risk assessment applies to complex processes potentially accounting for adverse events and reactions that undergo well-established legislative oversight, and nonconformances (NCs), that lack of established tracking systems. NCs account for an integral part of the quality management system, so that their documentation is important. The study evaluated the performance of a customized tool for incident reporting (IR) to track and characterize NCs in a public IVF center. IVF operators used the IVF-customized IR tool to record NCs at the moment of detection or subsequently, and in a time-saving manner during daily practice. From February 2015 to February 2020, 635 NCs were reported leading to the implementation of 10 operative instructions and 3 procedures with corrective strategies. NCs referred to the IVF laboratory were the most numerically meaningful (454/635, 71.5%). The majority (352/454, 77.5%) accounted for NCs related to procedures of sample management; considering the analytical phase as all the procedures involving sample treatment, the intra-analytical phase (176/352, 50%) has always been more subject to NCs compared to pre- (102/352, 29%) and postanalytical (74/352, 21%) phases. Our experience showed that the IVF-customized IR tool is suitable for application in IVF with regard to NC reports and documentation, as it identifies the most vulnerable steps of treatments. It manages NCs over the time, but it requires a contextual understanding of its application in order to avoid NC underestimates that could negatively influence the safety and quality aspects of IVF treatments.

Background The worldwide spread of the SARS‐CoV‐2 infection has profoundly affected all aspects of human life, with tangible consequences in several contexts, including reproduction. However, evidences on the inter‐relation between psychological distress and reproductive medicine are still conflicting. Methods The national lockdown imposed in Italy in March‐May 2020 and the consequent assisted reproductive techniques (ART) activity blockage allowed to evaluate the conception ability of couples who suffered the postponement of ART cycles. In particular, we collected anamnestic, anthropometrical and demographic data of those women attending ART straddling the lockdown period attending to the Fertility Centre of Reggio Emilia. Results Among the 431 couples recalled to reschedule ART cycles, 34 couples (7.9%) obtained a spontaneous pregnancy during the lockdown. Comparing spontaneously pregnant to non‐pregnant women, the pregnant group resulted younger (P = 0.009) and with a shorter infertility history (P = 0.029). Interestingly, the sexual activity frequency was significantly higher in pregnant women compared to non‐pregnant ones (P

The predictive role of sperm motility and morphology was recently detected in a large sample of more than 20000 assisted reproductive technology (ART) fresh cycles. However, the complete ART procedure consisted of both fresh and frozen-embryos transfers and only a comprehensive evaluation of the entire process could really confirm if these parameters really predict the ART success. The aim of the study was to evaluate which sperm parameter could predict the success of ART.A retrospective, real-world data analysis was performed, enrolling all couples attending ART from 2008 to 2021, including both fresh and frozen cycles and both in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI) procedures.Fresh cycles success (considering live birth rate) was predicted by female age (1.04 [1.02-1.06]), injected oocytes (0.96 [0.93-0.99]), embryo number (0.79 [0.75-0.83]) and progressive sperm motility (0.98 [0.97-0.99]). On the contrary, frozen cycle outcomes were predicted only by sperm motility (0.97 [0.95-0.99]). This prediction was confirmed in IVF but not in ICSI cycles.Both female and male parameters predicted the ART success considering the entire path. However, frozen cycle success was predicted only by progressive sperm motility in IVF cycles, suggesting that the potential amelioration of this male parameter is relevant to improve ART success. Those couples expected to obtain the highest embryos after fertilization (low female age and better semen parameters) will have more attempts with frozen cycles and thus would benefit from a potential treatment focused to improve sperm parameters.

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

Study question Evaluation of the effect of varicocele correction on conventional and microscopic seminal parameters and evaluation of which factors might predict sperm improvement after surgical treatment. Summary answer The detailed morphologic sperm evaluation has been identified as a novel parameter expressing the post-surgical semen amelioration after varicocelectomy. What is known already Generally, varicocele develops during puberty and occurs more often on the left side for anatomic reasons. However, its diagnosis is commonly delayed, especially in asymptomatic cases, until the man consults an andrologist for couple infertility. A causative relationship between varicocele and impairment of semen quality has been largely investigated in the context of male infertility. Despite the florid literature available on this topic, the clinical benefit in terms of semen quality improvement after varicocele surgical repair remains controversial. Study design, size, duration An observational, retrospective clinical trial was carried out including patients undergoing surgical treatment for varicocele at the Day Surgery of the Urology Operative Unit of the Santa Maria Nuova Hospital-IRCCS of Reggio Emilia from September 2011 to March 2020. Primary outcome was the detailed morphologic microscopic sperm evaluation. Secondary outcomes were conventional semen analyses. Each patient was considered two times (before and after the surgery) and evaluated by both physical examination and ultrasonography. Participants/materials, setting, methods The inclusion criteria considered the patients with diagnosis of varicocele at ultrasound examination, attending surgical resolution and with at semen analyses before and after the surgery, were excluded patients with diagnosis of varicocele without surgical indication, and/or semen analyses performed only before or only after the surgery. In the statistical analysis a logistic multivariate regression analysis was performed in order to evaluate the change before and after surgery. Main results and the role of chance The post-surgical semen analysis was performed after a mean of 183.7 + 112.5 days since the surgery for a total of 121 males (mean age 24.6 + 4.1 years) enrolled. The surgical treatment leads to a significant increase in sperm concentration (p = 0.015) and percentage of progressive and total motility (p = 0.022 and p = 0.039, respectively), with a significant decrease in the percentage of immobile sperms (p = 0.013). In particular, semen concentration improved in 71.7% of patients (p = 0.010). Considering the detailed morphologic microscopic evaluation, a significant improvement was detected: head abnormalities showed a significant reduction, considering microcephaly (3.3 + 3.6 versus 2.2 + 2.9%, p = 0.015), macrocephaly (1.4 + 0.6 versus 1.2 + 0.9%, p = 0.043) and cytoplasmic appendix (1.4 + 0.8 versus 0.9 + 1.2%, p = 0.041). Moreover, surgery led to a significant reduction of tails abnormalities, considering absence (0.6 +2.3 versus 0.1 + 0.7, p = 0.048) and coiled tail (5.2 + 1.5 versus 6.6 + 2.0, p = 0.037). Thus, surgical varicocele resolution leads to a significant improvement in specific morphological semen parameters. The multivariate logistic analysis identified the ultrasound varicocele degree before surgery as a main predictor of the sperm concentration improvement (p = 0.016). The semen parameters improvement was higher for varicocele of I and II degree (p = 0.008). Limitations, reasons for caution The retrospective study design precluded from carrying out a case-control study to compare the surgical techniques. Moreover, the study design limited the availability of patients’ clinical data in order to performed a more comprehensive predictive analysis. Wider implications of the findings Using a complex statistical approach, it emerged that the greatest improvement in semen quality was obtained in case of mild varicocele, increasing the knowledge on the therapeutic potential of surgery. This result has clinical implications, since it could help to select those patients ‘to treat or not to treat’. Trial registration number none

Study question Evaluate the population attending Assisted reproductive techniques (ART) who have suffered the interruption of fertilization paths because of the SARS-CoV–2 italian national lockdown declared under goverment provision. Summary answer The research show that some infertile couples candidate to ART paths obtained a spontaneous pregnancy during the lockdown due to SARS-CoV–2 pandemic. What is known already The real impact of psychological stress on couple infertility in terms of pregnancies obtained is largely under-investigated in the literature and the potential low frequency of sexual activity is usually poorly considered in the management of couple infertility and its role on pregnancies failure is unclear, but probably underestimated. Moreover, the worldwide spread of the SARS-CoV–2 infection has profoundly affected all aspects of human life, with tangible consequences in several contexts, including reproduction. This allowed to highlight the interrelation between psychological distress and reproductive medicine are still conflicting. Study design, size, duration This is a study conducted at the Fertility Centre of the Department of Obstetrics and Gynaecology of Reggio Emilia (Italy), that evaluated the conception ability of couples who suffered the postponement of ART cycles during the SARS-CoV–2 pandemic. In particular, we collected anamnestic, anthropometrical and demographic data of those women attending ART straddling the lockdown period, from March 2020 to April 2020. Participants/materials, setting, methods The study evaluated couples attending ART, who had an interruption of the fertilization paths due to the SARS-CoV–2 pandemic. The variables as women age, BMI, duration of infertility, number of sexual intercourses per week and infertility aetiology were considered in a multivariate logistic analysis. The statistical analysis was performed setting pregnancy (categorical data) as the dependent variable, and all other available data as either covariates or cofactors. Main results and the role of chance Among the 431 couples recalled to reschedule ART cycles 34 couples (7.9%) obtained a spontaneous pregnancy during the COVID–19 lockdown. The statistical analysis for the 431 couples recalled showed that average duration of couple infertility was 3.5 ± 2.8 years, while an exclusively female causal factor was observed in the 23.7% of cases (102 couples), an exclusively male one in the 32.7% (141 couples), a coexistence of male/female factor in the 18.6% (80 couples), and an idiopathic form in the 25.1% (108 couples). The 34 couples obtained a spontaneous pregnancy presented a female infertility factor in the 26.5% of cases (9 couples), a male factor in the 29.4% (10 couples), a male/female factor in the 11.8% (4 couples), and idiopathic infertility in the 32.4% (11 couples). The male factor of infertility was not evaluated in all couples, thus this definition came from the anamnestic evaluation of the couple. In a multivariate logistic analysis we highlighted that the infertility history duration and the sexual intercourses frequency were significantly related to pregnancy (F = 4.8, degrees of freedom=1, p = 0.030 and F = 81.6, degrees of freedom=1, p Limitations, reasons for caution Despite the short observation period (two months of italian national lockdown declared under goverment provision), the large sample size of women evaluated attending to a single ART centre constitutes a point of strength of our study. However, the absence of a control group represents the most important limit. Wider implications of the findings: In conclusion, the lockdown allowed to increase the knowledge about under-explored causes of idiopathic infertility: the frequency of sexual intercourses. This aspect in reproductive medicine could help to identify those couples able to conceive spontaneously, avoiding unnecessary over-treatment, and to correctly apply ART to those couples who really need it. Trial registration number none

Aim of this study was to investigate whether parental mental representations during pregnancy and after delivery differed between parents who conceived after Assisted Reproductive Treatments (ART) and spontaneous conceiving (SC) parents. Effects of specific ART variables (previous ART attempts, treatment type and cause of infertility) were also taken into account. Seventeen ART couples and 25 SC couples were recruited at Santa Maria Nuova Hospital (Reggio Emilia, Italy). At both 32 weeks of gestation (T1) and 3 months postpartum (T2) participants completed the Semantic Differential of the IRMAG, a self-report tool which measures specific domains of mental representations pertaining either individual (Child, Self-as-woman/man, and Partner) or parental (Self-as-parent, Own parent) characteristics. Results showed that ART parents had significantly more positive representations of the child compared to SC parents, while the scores at Partner dimension improved from T1 to T2 for SC parents only. With regards to ART history, scores at the Self-as-woman/man dimension were significantly less positive for ICSI than IVF parents and improved substantially from T1 to T2 only in case of mothers with previous ART attempts and of fathers at the first ART cycle. The representation of own parents increased from T1 to T2 in case of infertility diagnosis due to male factors, while a decrease emerged when infertility was due to female factors. Findings suggest the need to investigate parental mental representations after ART, in order to improve the understanding on the transition to parenthood of infertile couples and to target more specific intervention for parenting support.

The impact of the COVID-19 pandemic on global healthcare workers’ (HCWs) mental health has been well documented in the last two years; however, little is known regarding HCWs working in specific healthcare fields. During two subsequent periods of national lockdown in Italy (June–July 2020, T1, and November–December 2020, T2), a total sample of 47 HCWs working in a reproductive medicine hospital unit completed an ad hoc questionnaire for assessing emotional reactions to the pandemic, stress symptoms, and ways of coping. Moderate–high levels of anger and sadness were experienced by 65.9% and 68.1% of the HCWs, respectively, while moderate–high levels of anxiety and fear were experienced by 51.1% and 56.8%, respectively. Higher stress symptoms experienced by HCWs were hypervigilance, avoidance of thoughts and memories, and tiredness/low energy. At T2, levels of hypervigilance, irritability, intrusive thoughts, and detachment were higher than at T1, while avoidance of external triggers decreased. Moderate–high levels of anxiety resulted significantly associated with several symptoms of stress: irritability/fearfulness, depression/hopelessness, tiredness/low energy, problems with concentration, and intrusive thoughts. Regarding coping strategies, HCWs tended to adopt more problem-focused coping (e.g., contributing to improving a situation) and this tendency was higher at T2. Overall findings suggest a risk for the persistence of stress symptoms and, therefore, a risk for a chronic course, which might interfere with the global quality of mental health at work and the care provided to patients. Clinical implications highlight the relevance of implementing support programs for this category of HCWs focused on the elaboration of negative emotions and on fostering adaptive coping strategies.

Setting: A causative relationship between varicocele and impairment of semen quality has been largely investigated in the context of male infertility, although its clinical benefit remains controversial.Objective: To investigate the effect of varicocele correction on detailed morphologic microscopic semen parameters in a large homogeneous cohort of patients and to evaluate which factors could predict semen improvement after the surgical treatment.Design: An observational, retrospective cohort study was carried out including all patients undergoing surgical treatment for varicocele from September 2011 to March 2020 in the same clinical centre. Enrolled males performed at least one semen analysis before and one after surgical varicocele correction. Primary outcome was the detailed morphologic microscopic sperm evaluation. Secondary outcomes were conventional semen analyses.Results: 121 males (mean age 24.6+6.1 years) were enrolled. Using detailed morphologic microscopic sperm evaluation, a significant morphological improvement was recorded, with a reduction in head and tail abnormalities. Moreover, a significant increase in sperm concentration (p=0.015) and percentage of progressive and total motility (p=0.022 and p=0.039) were observed after surgery. The multivariate logistic analysis identified the ultrasonography varicocele degree before surgery as a main predictor of the sperm concentration improvement (p=0.016), with the highest improvement for varicocele of I and II degree. Conclusion: For the first time, the detailed morphologic microscopic sperm evaluation highlights a relevant reduction in sperm abnormalities after varicocele surgery, showing its potential application in clinical practice.

Background Quantitative real time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) are methods used for gene expression analysis in several contexts, including reproductive endocrinology. Objectives Herein, we compared qPCR and ddPCR technologies for gene expression analysis of hormone membrane receptor-encoding genes (FSHR, GPER and LHCGR), as well as the commonly used RPS7 housekeeping gene, in order to identify the most reliable method to be applied for gene expression analysis in the context of human reproduction. Methods Total RNA was extracted from human primary granulosa lutein cells of donor patients undergoing assisted reproduction and used for gene expression analysis by qPCR and ddPCR, after finding the optimal annealing temperature. Immunostaining for protein localization in cell membranes was also performed. Results Both techniques provided results reflecting the low number of FSHR and GPER transcripts, although ddPCR quantified the low-expressed genes with major accuracy, thanks to its higher reaction efficiency. The absolute FSHR and GPER transcript number was also determined by ddPCR, resulting in 40- to 260-fold lower amount than LHCGR transcripts. qPCR and ddPCR data are convergent with immunofluorescence analysis of membrane receptor expression in human primary granulosa lutein cells. Conclusion These results suggest that ddPCR is the candidate technology for analysis of genes with relatively low expression levels and provides useful insights for characterizing hormone receptor expression levels in the context of reproductive endocrinology.

Objective: To appraise the fertilization rate as a predictive factor for cumulative live birth rate (CLBR). Design: Multicenter retrospective cohort study. Setting: Ten in vitro fertilization clinics, whose data were collected and processed by the assisted reproductive technology (ART) Italian National Registry. Patient(s): 7,968 couples undergoing 9,394 complete intracytoplasmic sperm injection cycles. Intervention(s): None. Main Outcome Measure(s): The primary outcome measure was the CLBR in association with the fertilization rate intervals (80%—group 3). Further data stratification was performed on the basis of maternal age ( 10 oocytes). Result(s): The CLBR was progressively higher in relation to the fertilization rate in groups 1, 2, and 3 (20.1%, 34.7%, and 41.3%, respectively). The number of recovered oocytes, embryo number per cycle, and cumulative pregnancy rate followed the same trend. The decrease in CLBR with increasing maternal age was significantly correlated with the fertilization rate and CLBR in all 3 maternal age groups. Multivariate logistic regression analysis showed fertilization rate as a factor independently associated with CLBR. Conclusion(s): The present data indicated a positive association between the fertilization rate and the CLBR, suggesting the predictive clinical relevance of this parameter and its adoption as a key performance indicator.

Commercial hMG drugs are marketed for the treatment of infertility and consist of highly purified hormones acting on receptors expressed in target gonadal cells. Menopur® and Meriofert® are combined preparation of FSH and hCG and are compared in vitro herein. To this purpose, the molecular composition of the two drugs was analyzed by immunoassay. The formation of FSH receptor and LH/hCG receptor (FSHR; LHCGR) heteromer, intracellular Ca2+ and cAMP activation, β-arrestin 2 recruitment and the synthesis of progesterone and estradiol were evaluated in transfected HEK293 and human primary granulosa lutein cells treated by drugs administered within the pg-mg/ml concentration range. Molecular characterization revealed that Meriofert® has a higher FSH:hCG ratio than Menopur® which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert® had a higher potency than Menopur® in inducing a cAMP increase. Moreover, Meriofert® revealed a higher potency than Menopur® in recruiting β-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-β-arrestin 2 complexes, as evidenced by a decrease in bioluminescence resonance energy transfer signal. This drug-specific activation of intracellular signaling pathways is consistent with the molecular composition of these preparations and impacts downstream progesterone and estradiol production, with Menopur® more potent than Meriofert® in inducing the synthesis of both the steroids. These findings are suggestive of distinct in-vivo activities of these preparations, but require cautious interpretation and further validation from clinical studies.

BackgroundQuantitative real time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) are methods used for gene expression analysis in several contexts, including reproductive endocrinology.ObjectivesHerein, we compared qPCR and ddPCR technologies for gene expression analysis of hormone membrane receptor-encoding genes, such as follicle-stimulating hormone (FSHR), G protein-coupled estrogen (GPER) and choriogonadotropin receptors (LHCGR), as well as the commonly used RPS7 housekeeping gene, in order to identify the most reliable method to be applied for gene expression analysis in the context of human reproduction.MethodsTotal RNA was extracted from human primary granulosa cells of donor patients undergoing assisted reproduction and used for gene expression analysis by qPCR and ddPCR, after finding the optimal annealing temperature.ResultsBoth techniques provided results reflecting the low number of FSHR and GPER transcripts, although ddPCR detected also unspecific transcripts in using RPS7 primers and quantified the low-expressed genes with major accuracy, thanks to its higher reaction efficiency. The absolute FSHR and GPER transcript number was also determined by ddPCR, resulting in 50- to 170-fold lower amount than LHCGR transcripts.ConclusionThese results suggest that ddPCR is the candidate technology for analysis of genes with relatively low expression levels and provides useful insights for characterizing hormone receptor expression levels in the context of reproductive endocrinology.

Mechanisms regulating the selection of antral ovarian follicles are poorly understood and supposed to rely on low estrogen levels, decline of follicle-stimulating hormone (FSH) levels and receptor (FSHR) expression. These concepts are challenged in vitro, where apoptosis of human granulosa cells (hGLC) and transfected cell lines is induced by high doses of FSH or FSHR overexpression, while estrogens induce anti-apoptotic signals via nuclears and a G protein-coupled estrogen receptor (GPER). Therefore, in vitro data suggest that antral follicle selection may be driven by underestimated, FSH/FSHR-dependent apoptotic signals due to transiently maximized FSHR expression and overload of cAMP signalling, prevailing on estrogen-dependent signals. Here we demonstrate how FSHR/GPER physical interaction rescue ovarian follicles from FSH-mediated death. 10 nM FSH induces high intracellular levels of cAMP, measured by bioluminescence resonance energy transfer (BRET), and apoptosis in cultured hGLC under conditions where GPER levels are depleted by siRNA. This result was confirmed in transfected HEK293 cells overexpressing FSHR. Using BRET, photo-activated localization microscopy (PALM) and bioinformatics prodiction, we also demonstrate FSHR/GPER heteromers at the cell surface. The role of FSHR/GPER heteromers may be relevant to inhibit FSH-induced death signals, since increasing GPER expression levels in HEK293 cells co-expressing FSHR results in displacement of the Gαs-protein to FSHR, blockade of FSH-induced cAMP production and inhibition of apoptosis. However, in HEK293 cells coexpressing GPER/FSHR, FSH-induced activation of the anti-apoptotic AKT-pathway via a Gβγ-dependent mechanism, as demonstrated by Western blotting in cells treated using the inhibitor gallein. Inhibition of both FSH-induced cAMP production and apoptosis was lost when FSHR is coexpressed together with a mutant GPER, unable to heteromerize with FSHR, as well as in KO HEK293 cells unable to produce a molecular complex associated with GPER inhibiting cAMP. GPER/FSHR coexpression is confirmed in secondary follicles from paraffin-embedded tissues of human ovary by immunohistochemistry, suggesting that FSHR-GPER heterodimers could be physiologically relevant in vivo for inhibiting cAMP-linked apoptosis. Most importantly, FSHR and GPER co-expression correlates in hGLC from FSH-normo-responder women undergoing assisted reproduction, while it is not in hGLC from FSH-poor-responders, where increasing FSHR mRNA levels do not correspond to increasing GPER mRNA levels. We demonstrate that death signals in atretic follicles are delivered through overexpressed FSHR and inhibited by FSHR/GPER heteromerization, activating anti-apoptotic pathways. This finding unveils a novel working model of the physiology of dominant follicle selection and the relationship between FSH and estrogens.

Classically, follicle stimulating hormone receptor (FSHR) driven cAMP-mediated signaling boosts human ovarian follicle growth and would be essential for oocyte maturation. However, contradicting in vitro suggest a different view on physiological and clinical significance of FSHR-mediated cAMP signaling. We found that the G protein coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are effectively delivered upon equal expression levels of both receptors, while they are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with FSH responsiveness of patients undergoing controlled ovarian stimulation. Consistent with high FSHR expression levels during follicular selection, cell viability is dramatically reduced in FSHR overexpressing cells due to preferential coupling to the Gαs protein/cAMP pathway. In contrast, FSHR/GPER heteromer formation resulted in FSH-triggered anti-apoptotic/proliferative signaling delivered via the Gβγ dimer while heteromer impairment or GPER-associated Gαs inhibitory protein complexes resulted in cell death. GPER-depleted granulosa cells have an amplified FSH-dependent decrease in cell viability and steroidogenesis, consistent with the requirement of estrogen signaling for successful oocyte growth. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.One Sentence SummaryFSHR/GPER heteromers block cAMP-dependent selection of ovarian follicles and target tumor growth and poor FSH-response in women.

This study aims to describe parents&rsquo, and infant&rsquo, s interactive styles after assisted reproduction treatments (ART), to compare them with parent&ndash, infant interactions after spontaneous conception (SC), and to assess the effect of specific ART variables (cause of infertility, treatment type, and previous ART attempts) on interaction quality. The sample included 25 ART conceiving couples and 31 SC couples with their 3-months-old babies. Free parent&ndash, infant interactions (3&ndash, 5 min) were coded using the CARE-Index, a video-based assessment scale that gives both dimensional (e.g., sensitivity, control, passivity) and categorical scores (sensitive, inept, at-risk) for parents and infants. Results showed a global similarity between groups in CARE-Index dimensions. Nevertheless, differences emerged in categorical scores, as the interactive patterns of ART parents were more frequently classified as &ldquo, inept&rdquo, and &ldquo, at-risk&rdquo, compared to SC parents. With regards to ART dyads only, infants conceived through intracytoplasmic sperm injection scored significantly lower to the dimension compulsivity and higher to passivity, compared to infants conceived through in vitro fertilization. Yet, infants conceived at the first ART cycle had significantly lower levels of difficulty than infants conceived after one ART attempt. These results speak about the existence of important parent&ndash, infant interactive differences related to conception modality and ART technique and suggest the need to implement support programs to promote more sensitive parenting styles.

Background In the absence of international guidelines indicating the usage of vitrification rather than slow-freezing, the study aim was to analyze a large cohort of slow-frozen/thawed embryos to produce a rationale supporting the standardization of IVF cryopreservation policy. Methods This retrospective analysis included 4779 cleavage stage embryos cryopreserved by slow-freezing/thawing from September 2009 to April 2017 at a single Center. Biological and clinical outcomes of three different commercial kits adopted sequentially, i.e. Vitrolife Cleave Kit® from Vitrolife (kit 1) vs. K-SICS-5000 Kit® and K-SITS-5000 Kit® from Cook Medical (kit 2) and Freeze/Thaw 1™ Kit® from Vitrolife (kit 3) were collected and compared in the light of cryoprotectants composition. Results Kit 3 compared to kit 1 and kit 2 showed significantly (P P P P Conclusions A defined concentration of extracellular cryoprotectants in thawing/warming solutions had a beneficial effect on the embryo cryosurvival rate. Results could provide the rationale for the adoption of a single standardized warming protocol.

Background and aims Sphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid. The role of the lysosphingolipid in gonads of the female is widely unclear. At nanomolar concentrations, S1P binds and activates five specific G protein-coupled receptors (GPCRs), known as S1P1-5, modulating different signaling pathways. S1P1 and S1P3 are highly expressed in human primary granulosa lutein cells (hGLC), as well as in the immortalized human primary granulosa cell line hGL5. In this study, we evaluated the signaling cascade activated by S1P and its synthetic analogues in hGLC and hGL5 cells, exploring the biological relevance of S1PR-stimulation in this context. METHODS AND RESULTS. hGLC and hGL5 cells were treated with a fixed dose (0.1 μM) of S1P, or by S1P1- and S1P3-specific agonists SEW2871 and CYM5541. In granulosa cells, S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced CREB phosphorylation. No cAMP production was detected and pCREB activation occurred even in the presence of the PKA inhibitor H-89. Moreover, S1P-dependent CREB phosphorylation was dampened by the mitogen-activate protein kinase (MEK) inhibitor U0126 and by the L-type Ca2+ channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P2 or S1P3 with the specific receptor antagonists JTE-013 and TY52156, or under PLC/PI3K depletion. S1P-dependent CREB phosphorylation induced FOXO1 and the EGF-like epiregulin-encoding gene (EREG), confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. However, S1P or agonists did not modulate granulosa cell viability and proliferation in our conditions. Conclusions This study demonstrates for the first time that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, although this is not sufficient to induce intracellular steroidogenic signals and progesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P–S1PR axis may cooperate with gonadotropins in modulating follicle development.

This study assessed the relationship between regular physical activity and reproductive performance in obese infertile patients who receive assisted reproduction cycles with stable bodyweight. A total of 216 obese infertile women at their first fresh assisted reproduction attempt with stable body mass index (BMI) and available data on their physical activity carried out up to the beginning of the treatment cycle were enrolled in this observational cohort study. Clinical and biological data were recorded and analysed. There were 41 obese patients who did regular physical activity and 175 obese controls who did not. Total pregnancies (16/41, 39.0% versus 28/175, 16.0%, respectively; P = 0.002) and live births (10/41, 24.4% versus 13/175, 7.4%, respectively; P = 0.004) were significantly higher in patients who did physical activity regularly compared with those who did not. After adjusting for confounders, in obese infertile patients who did physical activity regularly, the relative risks for a clinical pregnancy and live birth were 3.22 (95% CI 1.53-6.78; P = 0.002) and 3.71 (95% CI 1.51-9.11; P = 0.004), respectively. In conclusion, regular physical activity carried out before a assisted reproduction cycle is significantly related with improved reproductive performance in obese infertile patients, irrespective of bodyweight loss. Body weight loss improves not only spontaneous pregnancy rates but also those of assisted reproductive techniques (ARTs). Moreover, almost all studies refer to body weight loss due to lifestyle intervention programs consisting in hypocaloric diet and increased physical activity. Instead, very little is known about the specific effects of physical activity alone on human reproduction. Based on these considerations, we designed the present study to assess the relationship between regular physical activity and reproductive outcome in infertile obese patients who receive ARTs. Two-hundred-sixteen obese infertile women with stable body mass index (BMI) and at their first fresh ART attempt were enrolled, and clinical and biological data were recorded and analyzed. Our results demonstrate that the chances to obtain a pregnancy and a baby are 3-fold higher in obese infertile patients who does physical activity regularly in comparison with those who does not, suggesting that regular physical activity before ART cycles improves the reproductive performance in obese women irrespective to body weight loss.

Objective To evaluate the effect of the 2004 Italian regulations (insemination of ≤3 oocytes/cycle, transfer of all embryos, prohibition of embryo cryopreservation) on outcomes of assisted reproduction treatment (ART). Design Case-control study. Setting The Center of Reproductive Medicine, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy. Patient(s) Women undergoing ART for the first time. Intervention(s) Comparing outcomes of ART between 2 years before (n = 900) and after (n = 936) the law's implementation (March 10, 2004). Main Outcome Measure(s) Rates of fertilization, pregnancy, "take-home baby," and multiple pregnancies. Result(s) During the pre-law period, statistically significantly more patients reached embryo transfer (odds ratio 1.9; 95% CI, 1.5, 2.5), and embryo transfer rate per cycle was statistically significantly higher (3.1 ± 1.7 vs. 2.2 ± 0.7), but the overall transfer of good embryos was lower (OR 0.6; 95% CI, 0.5, 0.8). The pregnancy rates per aspiration cycle were similar between the periods, but the pregnancy rate per embryo transfer and birth rate with at least one liveborn baby per embryo transfer were statistically significantly lower in the pre-law period (OR 0.7; 95% CI, 0.5, 0.9). The multiple births rate was not different between the two periods. Conclusion(s) In contrast to prior pessimistic expectations, the obligation to transfer all available embryos produced from ≤3 inseminated oocytes neither reduced success rates of ART nor increased the multiple births rate.

Background Intracytoplasmic morphologically selected sperm injection (IMSI) is still proposed and employed in the clinical practice to improve the reproductive outcome in infertile couples scheduled for conventional intracytoplasmic sperm injection (cICSI). The aim of the current randomized controlled trial (RCT) was to test the hypothesis that IMSI gives a better live birth delivery rate than cICSI.

Purpose: To determine whether maternal age and number of transferred embryos influence early pregnancy losses in twin pregnancies compared to singletons following IVF/ICSI. Methods: We compared the pregnancy loss rates in singleton (n = 549) and twin (n = 252) gestations, stratified by maternal age (≤35 and > 35 years) and the number of transferred embryos (1–3 and 4–9). Results: Loss rates of singleton pregnancies were significantly higher than that in twins (OR 3.0, 95% CI 1.9, 4.9), especially among singletons conceived after transfer of 4–9 embryos (OR 5.0, 95% CI 2.2, 11.9). Younger mothers of twins had lower loss rates (OR 0.3, 95% CI 0.1, 0.9). Conclusion: Twins have a significantly reduced spontaneous miscarriage rate compared with singletons following IVF/ICSI. Higher implantation rates per cycle (i.e., development of twins rather than one live embryo) may represent a better capacity of the uterus for early embryonic development.

To determine whether pregnancies after IVF, with and without intracytoplasmic sperm injection (ICSI), have different early spontaneous loss rates.Retrospective analysis of IVF/ICSI dataset.The Center of Reproductive Medicine, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy.Women undergoing IVF with or without ICSI.First-trimester sonography at 6-7 weeks to count the number of embryos with positive heartbeat. The number of embryos lost was calculated from a second-trimester sonogram.Embryonic loss rates related to the initial number of embryos, maternal age35 oror =35 years, and IVF procedure.In vitro fertilization and ICSI had similar embryonic loss rates (odds ratio [OR] 1.2, 95% confidence interval [CI] 0.9-1.7, and OR 1.3, 95% CI 0.9-1.8 for women aged35 years andor =35 years, respectively). Younger women had fewer losses after IVF (OR 0.7, 95% CI 0.5-0.9). Multiples had lower loss rates compared with singleton pregnancies.In vitro fertilization and ICSI have similar spontaneous embryonic loss rates. Factors other than the initial number of embryos, maternal age, and IVF technique, such as embryo quality or uterine environment, might be involved in the outcome of multiple pregnancies in assisted reproductive technology procedures.

After the birth of more than 900 babies from human cryopreserved oocytes with no apparent increase in congenital anomalies compared to conventional IVF babies [1], oocyte cryopreservation has become a quite widespread technique in Assisted Reproductive Technologies (ART), even in the female fertility preservation programs. Today two procedures are available for oocyte cryopreservation: slow-freezing (SF), the first protocol introduced, and vitrification (VT) [2]. The main goal of VT is to achieve high cryoprotectant concentration in order to increase the viscosity of the cryoprotectant solution and to suppress ice nucleation [3]. Biological and clinical outcomes of SF protocols seem to be less than those obtained with fresh and vitrified oocytes [4–6]. Oocyte cryopreservation is usually used in ART laboratories for supernumerary oocyte storage after Controlled Ovarian Stimulation (COS) [4], prevention of Ovarian HyperStimulation Syndrome (OHSS) risk, oocyte accumulation in poor responder patients [7], and fertility preservation programmes for cancer patients [8–10] or for social reasons [11]. Nevertheless, in 2008 the Practice Committee of American Society of Reproductive Medicine (ASRM) concluded that “Oocyte cryopreservation presently should be considered an experimental technique,” as the data available on the efficacy of oocyte cryopreservation in relation to the length of storage are still limited [1,12]. Instead, the ASRM considers embryo cryopreservation a non-experimental technique [12], with a proven clinical outcome regardless of the length of storage [13,14]. Unfortunately, there are limited data concerning children born from long-term cryopreserved oocytes [1]. The first live birth obtained from oocytes stored for a long period was reported by Yang et al. in 2007: oocytes were cryopreserved by SF for fertility preservation before cancer treatment and thawed 6 years later . In 2008, Parmegiani et al. reported a live birth from oocytes cryopreserved by SF after 5 years of storage in a patient who underwent infertility treatment. Kim and Hong [17] recently described a similar result, reporting a live baby born from vitrified oocytes thawed 5 years later . Here we describe the case of a woman who underwent ART procedures for both female (tubal) and male (oligoastenotheratozoospermia) infertility in our non private sterility center. The patient gave birth to two babies in two different thawing cycles from cryopreserved oocytes with SF protocol: the first baby after 11 months and the second baby after 6 years of storage.

Intracytoplasmic morphologically selected sperm injection (IMSI) is still proposed and employed in the clinical practice to improve the reproductive outcome in infertile couples scheduled for conventional intracytoplasmic sperm injection (cICSI). The aim of the current randomized controlled trial (RCT) was to test the hypothesis that IMSI gives a better live birth delivery rate than cICSI. Infertile couples scheduled for their first cICSI cycle for male factor were allocated using a simple randomization procedure. All available biological and clinical data were recorded and analyzed in a triple-blind fashion. Our final analysis involved the first 121 patients (48 and 73 subjects for IMSI and cICSI arm, respectively) because the trial was stopped prematurely on the advice of the data safety and monitoring Committee because of concerns about IMSI efficacy at the first interim analysis. No significant difference between arms was detected in rates of clinical pregnancy per embryo transferred [11/34 (32.3 %) vs. 15/64 (23.4 %); odds ratio (OR) 1.56, 95 % (confidence interval) CI 0.62–3.93, P = 0.343] and of live birth delivery [9/48 (18.8 %) vs. 11/73 (15.1 %); OR 1.30, 95%CI 0.49–3.42, P = 0.594). Current data did not support the routine use of IMSI in the clinical practice for improving cICSI results in unselected infertile couples with male factor.

Survivors of the "vanishing" twin syndrome, which occurred in dichorionic twins only, and singletons that began as singletons, had similar mean gestational duration and birth weights, as well as similar frequencies of maternal and neonatal complications. This similarity persisted when conventional IVF and IVF plus ICSI cases were separately evaluated.

Background The assessment of the embryo quality is crucial to maintain an high pregnancy rate and to reduce the risk of multiple pregnancy. The evaluation of the pronuclear and nucleolar characteristics of human zygote have been proposed as an indicator of embryo development and chromosomal complement. The aim of the current study was to assess the role of pronuclear morphology evaluation in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI) cycles. Methods Retrospective clinical analysis on 755 non-elective transfers of only one embryo (ET). Embryo assessment was performed in days 1 and 2. Clinical and biological data were recorded and analyzed according to embryo and/or pronuclear morphology. Results Both pronuclear and embryo morphology were significantly related to clinical pregnancy and live-birth rates. No significant difference in clinical pregnancy and live-birth rates was detected when the pronuclear and embryo morphology assessments were combined. Embryo morphology and maternal age were the only independent predictors of favorable outcome by logistic regression analysis. Conclusions Pronuclear evaluation is effective to select the best zygotes if ET is performed at day 1, whereas it did not improve the clinical outcomes when combined with embryo morphology evaluation in day 2.

Objective To evaluate how the unique Italian fertility regulations (≤3 inseminated oocytes/cycle, transfer of all embryos, prohibition of embryo cryopreservation) affected outcomes of ART. Study design Case–control study from the Center of Reproductive Medicine, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy. We compared outcomes of ART patients between five years before (n = 1791) and five years after (n = 2474) the implementation of the law. Results The mean embryo transfer (ET) rate was 3.1 ± 2.1 and 1.7 ± 1.1 before and after the law. Significantly more ICSI procedures were performed in women above 35 years old during the post-law period. The ET rate was higher before (88.6%) than after (80.5%) the law (OR 1.9, 95% CI 1.6, 2.2) especially in women >37 years undergoing ICSI (88.2 vs. 76.1%; OR 2.3, 95% CI 1.3, 4.2). The clinical pregnancy rates were practically unchanged but the proportion of triplet births significantly decreased after the law (10.3 vs. 4.1%, OR 2.7, 95% CI 1.4, 5.0). Conclusion In contrast to interim analyses, we found that the statutory obligation to transfer all available embryos produced from up to three inseminated oocytes reduced the ET rates, especially in older women, and decreased the triplet births rate.

Background Embryos selection is crucial to maintain high performance in terms of pregnancy rate, reducing the risk of multiple pregnancy during IVF. Pronuclear and nucleolar characteristics have been proposed as an indicator of embryo development and chromosomal complement in humans, providing information about embryo viability. Methods To correlate the zygote-score with the maternal age and the outcome of pregnancy, we analyzed the pronuclear and nucleolar morphology, the polar body alignment and the zygote configuration in 459 clinical pregnancies obtained by IVF and ICSI in our public clinic in Reggio Emilia, Italy. We derived odds ratios (OR) and Corenfield's 95% confidence intervals (CI). Continuous variables were compared with Student's t-test; P lower than .05 was considered statistically significant. Results We observed a significant increase of "A" pronuclear morphology configuration in 38-41 years old patients in comparison to that lower than or equal to 32 years old and a significant decrease of "B" configuration in 38-41 years old patients in comparison to that lower than or equal to 32 and in comparison to that of 33-37 years old. Related to maternal age we found no significant differences in P1 and in P2 configuration. We found no correlation between zygote-score, embryo cleavage and embryo quality. Conclusions Our results confirm the limited clinical significance of zygote-score suggesting that it can not be associated with maternal age, embryo cleavage and embryo quality. The evaluation of embryo quality based on morphological parameters is probably more predictive than zygote-score.

The Italian law regulating assisted reproductive technologies that came into force in 2004 restricts the number of fertilized oocytes per cycle to three, obliges the subsequent transfer of all resulting embryos and prohibits the freezing of surplus embryos. This study evaluates the impact of the law on severe oligozoospermic, cryptozoospermic, obstructive azoospermic and non-obstructive azoospermic patients. Intracytoplasmic sperm injection outcomes of 1066 cycles performed in the 4years before the passing of the law were compared with 804 cycles performed in the 4years after the law came to pass. Globally, analysis of clinical and obstetric outcomes showed a significant decrease in terms of pregnancy and delivery rates per cycle (17.8% versus 10.9% and 14.2% versus 8.5%, respectively) and per embryo transfer (18.8% versus 13.8% and 15.0% versus 10.7%, respectively), and a significant drop in multiple deliveries (35.1% versus 17.6%) in the post-law period. Cryptozoospermic and azoospermic couples were affected by the Italian law more than severe oligozoospermic couples. The results showed that the Italian law limits the efficiency of assisted reproduction treatment in couples with severe male factor. It is hoped that the Italian assisted reproductive technologies law is altered as soon as possible, allowing the insemination of more than three oocytes.

SummaryPronuclear morphology seems to be an important predictive value of zygote development and integrity. In this study we want to evaluate the effect of insemination technique, male factor and oocyte cryopreservation on pronuclear morphology of zygotes derived from sibling oocytes in our Centre of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova, Reggio Emilia, Italy. Subjects (n = 190) were submitted to IVF cycles with non-frozen and frozen sibling oocytes. Morphological evaluations were assessed using zygote pronuclear morphology (pronuclei, nucleoli and axis) in four groups: Group 1: 144 zygotes from 85 conventional IVF cycles with non-frozen oocytes; Group 2: 164 zygotes from 85 intracytoplasmic sperm injection (ICSI) cycles with Group 1 patients' sibling frozen oocytes; Group 3: 221 zygotes from 123 ICSI cycles with non-frozen oocytes; Group 4: 197 zygotes from 123 ICSI cycles with Group 3 patients' sibling frozen oocytes. No differences between Group 1 and Group 2 were seen. Group 3 was statistically different from Group 4 in relation to the nucleolar morphology. Oocyte cryopreservation procedure modified the nucleolar morphology of zygotes only in the presence of poor semen quality.

OBJECTIVE To assess the perinatal outcomes of the first three years under the 2004 Italian reproductive legislation obligating transfer of all embryos resulting from insemination of < or =3 oocytes. STUDY DESIGN We compared the perinatal results of clinical assisted reproductive technology (ART) pregnancies during the three years following the new Italian legislation with the previous three years. RESULTS There were 583 and 571 clinical pregnancies during the respective periods. Before the law, the overall embryonic and fetal loss rates were significantly higher resulting in a significantly lower rate of live born infants and significantly fewer clinical pregnancies with at least one live born infant. Quadruplet and quintuplet pregnancies were entirely eliminated following the 2004 law but the neonatal mortality rate was not different between the two study periods. CONCLUSION The 2004 Italian infertility legislation led to improved quantitative and qualitative outcomes of ART.

Objective To evaluate the effect of selecting three best oocytes for insemination and transfer of all resultant embryos on outcomes of assisted reproductive technologies (ART). Study Design Comparative study. Setting The Center of Reproductive Medicine, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy. Patient(s) Women undergoing ART during the period March 10, 2004 to March 9, 2006. Intervention(s) Comparing outcomes of ART following transfers of 3 "excellent" (n = 267) embryos and cycles in which none of the embryos was considered "excellent quality" (n = 176). Main Outcome Measure(s) Rates of fertilization, pregnancy, "take-home baby," and multiple pregnancies. Results The average number of retrieved oocytes and the average number of inseminated oocytes of good quality were similar in both groups. Intracytoplasmic sperm injection was performed four to five times more often in cycles without "excellent quality" irrespective of the patient's age (odds ratio 4.6, 95% confidence interval 3.0, 7.1). The two groups had similar rates of implantation, total pregnancies/ET, clinical intrauterine pregnancy, singleton and multiple births, and "take-home" baby rate. This similarity persisted irrespective of the patient's age. Conclusion(s) The selection of oocytes for insemination and transfer of all available embryos irrespective of their quality did not change outcomes of ART.

We examined the efficacy and effect of HAART in HIV-1-infected men confronted with assisted fertilization procedures. We showed that HAART did not always reduce the HIV-1-RNA level in blood and semen compartments, and that a significant upward shift in mitochondrial DNA was observed in spermatozoa from a HAART-treated patient group compared with spermatozoa from HAART-untreated or HIV-1-uninfected groups (P < 0.001). These findings emphasize the negative role of HAART, but not of HIV-1 infection, in determining semen alterations

The new Italian law on assisted reproduction technology rules that no more than three oocytes can be fertilized at one time, and that all embryos obtained must be transferred to the maternal uterus simultaneously. The aim of the present study was to investigate the influence of the new law on spontaneous embryonic/fetal losses through comparison of data collected over an identical period of time: the first year of application of the new law compared with the same time period 1 year before (March 10, 2004 to March 9, 2005 versus March 10, 2003 to March 9, 2004). A total of 271 clinical pregnancies were analysed. In the post-law period, a significantly lower percentage of total spontaneous embryonic losses compared with the pre-law period, and a higher percentage of surviving embryos in singletons and twins was observed. In conclusion, the impact of the limitations imposed by the new legislation regulating assisted reproduction in Italy seems to exert positive effects on spontaneous embryonic loss both in singletons and multiple pregnancies. These findings are in contrast to international predictions on this issue and, in general, are counter-intuitive. This suggests that further investigations on a larger cohort of women are required to confirm these preliminary results.

Objective To document outcomes of oocyte cryopreservation performed as a routine procedure in an IVF program. Design Describing the rate of oocyte survival, embryo transfer (ET), implantation, and live births of IVF–intracytoplasmic sperm injection performed on thawed oocytes. Setting Reproductive medicine center in Italy. Patient(s) Women (n = 696) who failed to conceive after IVF–intracytoplasmic sperm injection with fresh oocytes. Intervention(s) Surplus oocytes obtained during a failed cycle with fresh oocytes were frozen and then were thawed, micromanipulated, and transferred in a later cycle. Main Outcome Measure(s) Rates of oocyte survival, ETs, implantation, and live births were calculated in the entire cohort and in patients aged ≤38 and >38 years. Result(s) There were 29 pregnancies, for a total implantation rate of 6.3% (95% CI: 4.3, 9.0) per 456 ET cycles. The clinical-pregnancy rate was 19 (4.2%; 95% CI: 2.6, 6.4) of 456 ET cycles, with a take-home-baby rate of 7 (1.5%; 95% CI: 0.7, 3.0) of 456 ET cycles. Conclusion(s) Cryopreservation performed as a routine procedure for so-called salvaging of surplus oocytes is associated with poor implantation rates and with a probability of 1 live birth in 65 ET cycles.

Objective To determine whether embryo quality is associated with early spontaneous loss rates in twin and singleton pregnancies after IVF/intracytoplasmic sperm injection (ICSI). Design Retrospective, single center analysis. Setting The Center of Reproductive Medicine, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy. Patient(s) Women undergoing IVF/ICSI and two- or three-embryo transfer of intermediate- and top-quality embryos. Intervention(s) First-trimester sonography at 6 to 7 weeks to determine number of embryos with positive heartbeat. Number of embryos lost was calculated from a second-trimester sonogram. Main Outcome Measure(s) Rates of total pregnancy loss, as related to embryo quality, initial number of embryos, maternal age Results A total of 94 losses (23.1% of 407 pregnancies) were counted, with similar proportions in pregnancies after transfer of intermediate- or top-quality embryos. Neither the mode of IVF procedure nor the number of transferred embryos affected the loss rate. In contrast, the loss rate was significantly higher in older mothers after transfer of intermediate-quality embryos (odds ratio [OR 2.4], 95% confidence interval [CI] 1.1–5.5). Losses among singletons were significantly higher compared with losses among twins (OR 2.5, 95% CI 1.1–6.0), but this was observed in top-quality embryos only. Conclusion(s) Top-quality but not intermediate-quality ETs are associated with lower early spontaneous loss rates among twin pregnancies after IVF/ICSI.

Objectives The purpose of this study was to determine the early spontaneous loss rate in multiple pregnancies following assisted reproductive technology (ART). Study design Analysis of pregnancies following ART as related to the initial number of embryos and maternal age was performed. Results At least one spontaneous loss was observed in 47.0% (95% CI 29.8-64.9) of quadruplets, 59.3% (95% CI 48.2-69.8) of triplets, and 35.2% (95% CI 29.6-41.2) of twins. The increased loss rates in mothers ≥35 years was related to early twin pregnancies, but not to early triplet and quadruplet gestations. We could not find an effect of the starting number of embryos on either gestational age or birth weight characteristics. Conclusion Our observations did not identify a clear relation between early spontaneous loss of multiple gestations and either initial number of embryos or maternal age. Other factors might be involved in the outcome of ART multiple pregnancies.