First trimester pregnancy outcomes in a large IVF center from the Lombardy County (Italy) during the peak COVID-19 pandemic.

At the beginning of 2020, the Italian Lombardy region was hit by an "epidemic tsunami" which was, at that point in time, one of the worst pandemics ever. At that moment the effects of SARS-COV 2 were still unknown. To evaluate whether the pandemic has influenced ART (Assisted Reproduction Techniques) outcomes in an asymptomatic infertile population treated at one of the major COVID-19 epicentres during the weeks immediately preceding lockdown. All ART procedures performed during two time periods were compared: November 1st, 2018 to February 28th, 2019 (non-COVID-19 risk) and November 1st, 2019 to February 29th, 2020 (COVID-19 risk). In total 1749 fresh cycles (883 non-COVID-19 risk and 866 COVID-19 risk) and1166 embryos and 63 oocytes warming cycles (538 and 37 during non-COVID and 628 and 26 during COVID-19 risk, respectively) were analysed. Clinical pregnancies per cycle were not different: 370 (25.38%) in non-COVID versus 415 (27.30%) (p = 0.237) during COVID-19 risk. There were no differences in biochemical pregnancy rates 52 (3.57%) versus 38 (2.50%) (p = 0.089) nor in ectopic pregnancies 4 (1.08%) versus 3 (0.72%) (p = 0.594), spontaneous miscarriages 84 (22.70%) versus 103 (24.82%) p = 0.487, nor in intrauterine ongoing pregnancies 282 (76.22%) versus 309 (74.46%) p = 0.569. A multivariate analysis investigating differences in spontaneous miscarriage rate showed no differences between the two timeframes. Our results support no differences in asymptomatic infertile couples' ART outcomes between the pre COVID and COVID-19 periods in one of the earliest and most severe pandemic areas.

One step further: randomised single-centre trial comparing the direct and afterload techniques of embryo transfer.

STUDY QUESTION: What are the differences in ease of use between two different embryo transfer (ET) techniques: the preload direct approach and the afterload approach. SUMMARY ANSWER: The afterload technique seems to reduce the rate of difficult ETs. WHAT IS KNOWN ALREADY: Numerous published trials now document that the ET procedure has an impact on pregnancy and delivery rates after IVF. Difficult transfers should be avoided, as they reduce implantation and pregnancy rates. Preload direct ETs with soft catheters under ultrasound guidance is currently considered the best procedure. However, when using soft catheters, it is not known which technique is preferable or which one should be implemented to reduce the operator factor. STUDY DESIGN, SIZE, DURATION: This prospective randomised unblinded controlled clinical trial, included 352 ultrasound-guided ETs assigned to either direct ET or afterload ET, between September 2017 and March 2019. The sample size was calculated based on the historical rate of difficult ETs encountered between 2014 and 2015 with a direct ET procedure. PARTICIPANTS/MATERIALS, SETTING, METHODS: The inclusion criteria were women 18-38 years old, with BMI between 18 and 28, receiving a single-thawed blastocyst transfer. The exclusion criteria were use of testicular sperm and preimplantation genetic testing (PGT) cycles. The primary outcome was the rate of difficult or suboptimal transfers defined as: advancement of the outer sheath (specific for the direct transfer), multiple attempts, use of force, required manipulation, use of a stylet or tenaculum, dilatation, or use of a different catheter. The secondary outcome was clinical pregnancy rate. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 352 frozen ETs were randomised, with 176 patients in each group. The two arms were homogeneous for female and male age, female BMI, duration of infertility, secondary infertility, previous deliveries or miscarriages, myomas, previous surgery to the uterine cavity, cycle day at ovulation trigger, freeze all cycles, first transfers, indication for treatment, endometrial preparation protocol and duration, endometrial thickness, and blastocyst grade at vitrification. Across the entire population, 85 (24.1%) ETs were defined as difficult. The rate of difficult transfers was significantly higher in the direct ET group than in the afterload group: 68 (38.6%) versus 17 (9.7%), respectively (OR 0.17, 95% CI 0.09-0.30, P < 0.001). The mean percentage in the rate of difficult transfers per operator was 22.5% (SD ± 14.5%), of which 36.1% (SD ± 23.4%) were in the direct group compared with 8.6% (± 8.2%) in the afterload group (P < 0.001). The difficult transfer rate among operators varied from 0 to 43.8% (0-77.8% in the direct group and 0 to 25.0% in the afterload group). The clinical pregnancy rates (42.0% vs 48.3%, P = 0.239 in the direct and afterload groups, respectively) were not significantly different between the groups. LIMITATIONS, REASONS FOR CAUTION: There were 18 experienced operators who participated in the trial. Conclusions about the pregnancy rate should not be generalised, since the sample analysis was not performed on this outcome and, although clinically relevant, the difference was not significantly different. WIDER IMPLICATIONS OF THE FINDINGS: The rate of difficult transfers was significantly higher in the direct ET group compared with the afterload ET group, although a wide variation was observed among operators. Further studies regarding the association between transfer technique and ART outcomes are required. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was sought and there are no competing interests. TRIAL REGISTRATION NUMBER: NCT03161119. TRIAL REGISTRATION DATE: 5 April 2017. DATE OF FIRST PATIENT'S ENROLMENT: 26 September 2017.

The human factor: does the operator performing the embryo transfer significantly impact the cycle outcome?

STUDY QUESTION: Is Ongoing Pregnancy Rate (OPR) operator-dependent, and can experience improve embryo transfer efficiency? SUMMARY ANSWER: OPR is influenced by the operators who perform the embryo transfer (ET), and experience does not assure proficiency for everyone. WHAT IS KNOWN ALREADY: ET remains the critical step in assisted reproduction. Although many other factors such as embryo quality and uterine receptivity impact embryo implantation, the proper ET technique is clearly an operator-dependent variable and as such it should be objectively standardized. STUDY DESIGN, SIZE, DURATION: Retrospective comparative analysis including all fresh ETs performed between January 1996 and December 2016 at the Humanitas Fertility Center after IVF-ICSI cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: IVF/ICSI fresh ETs performed by 32 operators, 19 824 cycles in all, were analyzed. All transfers consisting of freehand insertion of a preloaded soft catheter into the uterine cavity under transabdominal ultrasound guidance were considered. Two different statistical analyses were performed. First, a logistic regression model with a random intercept for the operator was used to estimate the heterogeneity of the rate of success among operators, accounting for woman age, FSH, number of oocytes retrieved, fertilization rate, year of the procedure, number and stage of transferred embryos and operator's experience. Second, the relationship between experience and pregnancy rate was estimated separately for each operator by logistic regression, and operator-specific results were combined and compared in a random-effects meta-analysis. In both analyses, the operator's experience at time t was measured in terms of number of embryo transfers performed before t. MAIN RESULTS AND THE ROLE OF CHANCE: The heterogeneity among operators was highly significant (P value <0.001) and explained 44.5% of the total variability. The odds ratio of success of the worst operator in respect to the mean was equal to 0.84. For the best operator, the odds ratio of success was equal to 1.13 in respect to the mean. Based on the meta-analysis of the relationship between operator's experience and success rate, it resulted that, on average, the operators' performance did not improve with additional transfers. LIMITATIONS, REASONS FOR CAUTION: At our center, operators become independent for ET's after performing between 30 and 50 transfers under supervision. It is also possible that other relevant factors, such as embryologists on duty for the ET, have not been included in the present analysis and this may represent a potential bias. Among these, it should be mentioned that the embryologists on duty for the ET were not taken into consideration. WIDER IMPLICATIONS OF THE FINDINGS: Continued performance analysis and the use of a digital simulator could help operators to test their expertise over time and either correct poor performance or avoid doing transfers. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: NCT03561129.

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

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

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

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

Risk of cancer after assisted reproduction: a review of the available evidences and guidance to fertility counselors.

OBJECTIVE: Infertile women requiring ovarian stimulation and assisted reproduction techniques (ART) are faced with difficult issues. The fear that using hormones could increase their risk of cancer is the most significant. One of the main challenges for assessing cancer risk after ART is the difficulty to separate it from the underlying condition of infertility per se. The delay or the inability to achieve a pregnancy is an important risk factor for breast, endometrial and ovarian cancer. We analyzed the current literature on the topic. MATERIALS AND METHODS: The published literature in Medline and Cochrane was screened using the following keywords: ovulation induction, reproductive techniques, clomiphene, in vitro fertilization, fertility agents, female/adverse effects, female/toxicity gonadotropins/ adverse effects or gonadotropins/toxicity and "neoplasms or cancer". RESULTS: A total of 95 articles were evaluated. Limited evidence suggests that high doses or many cycles of clomiphene citrate could increase the risk of endometrial cancer, although the confounding factors of polycystic ovarian disease and overweight are not always considered. In some studies, ART modestly increased the risk of borderline ovarian cancer. Fertility treatments do not increase the risk of breast, cervical, endometrial and ovarian cancers, thyroid, melanoma and colon cancer. CONCLUSIONS: Women can be reassured that fertility drugs do not appear to significantly increase the risk of invasive ovarian, endometrial, breast or other cancers, while achieving a pregnancy at an earlier age is a significant protective factor.

Pro-inflammatory M1/Th1 type immune network and increased expression of TSG-6 in the eutopic endometrium from women with endometriosis.

OBJECTIVE: The study aimed to explore the type 1 and type 2 cytokines expression in the endometrium from women affected by endometriosis compared to controls. The expression of TSG-6, a multifunctional protein involved in several inflammatory disease, was also evaluated. Study Design SETTING: Experimental clinical study. PATIENTS: 10 patients affected by endometriosis and 11 controls. INTERVENTIONS: Patients underwent to an ultrasound transvaginal examination and a diagnostic hysteroscopy in order to exclude any uterine abnormality. All patients underwent endometrial biopsy using a Novak's curette. MAIN OUTCOME MEASURES: The endometrial expression of type 1 (IL- 1 ß TNF-α, IL-8) and type 2 (IL-10) cytokines, and of TSG-6 was evaluated by immunohistochemistry and by real time PCR. The expression of TSG-6 was confirmed by western blot. RESULTS: Results of PCR analysis and of immunohistochemistry revealed an increased expression of IL-1ß, TNF-α, IL-8 and of TSG-6 in the endometrium of endometriosic patients. IL-10 expression did not show any difference. CONCLUSIONS: An increased expression of pro-inflammatory type 1 cytokines was demonstrated in the endometrium from endometriosic patients, suggesting an endometrial environment harmful for implantation due to the prevalence of Th1 related immunity. An increased expression of TSG-6 was also demonstrated for the first time. Our findings concur to better define the inflammatory imbalance and the abnormal endometrial receptivity, reported in literature, of the eutopic endometrium of women affected by endometriosis.

Effects of pulsed electromagnetic fields on swelling and pain after implant surgery: a double-blind, randomized study.

The aim of this split-mouth, double-blind, randomized study was to determine whether pulsed electromagnetic field therapy (PEMF) can improve swelling and the management of pain after full-arch immediate loading implant surgery. Eleven patients were selected for the study. Each patient received four distal tilted implants in the upper or lower jaw and underwent full-arch immediate loading rehabilitation. After surgery, two PEMF devices were applied to each patient, one on each cheek. In a random manner, one of these PEMF devices was switched on (test side); the other served as a placebo (control side). Forty-eight hours after surgery clinicians estimated postoperative swelling through photographic documentation, comparing the condition before and after surgery, while pain was assessed using a verbal rating scale. The patient's degree of comfort in relation to the PEMF devices was analyzed by questionnaire using a numerical rating scale. No statistically significant difference was observed between the test and control sides for swelling or pain (P>0.05). Most of the patients did not present swelling or pain at 48h after surgery, regardless of whether the PEMF device was activated or not. Various outcomes were found in the comfort evaluation. Within the limitations of this study, PEMF does not reduce postoperative swelling or pain after implant surgery.

Human recombinant follicle stimulating hormone (rFSH) compared to urinary human menopausal gonadotropin (HMG) for ovarian stimulation in assisted reproduction: a literature review and cost evaluation.

BACKGROUND: Gonadotropins are protein hormones which are central to the complex endocrine system that regulates normal growth, sexual development, and reproductive function. There is still a lively debate on which type of gonadotropin medication should be used, either human menopausal gonadotropin or recombinant follicle-stimulating hormone. The objective of the study was to perform a systematic review of the recent literature to compare recombinant follicle-stimulating hormone to human menopausal gonadotropin with the aim to assess any differences in terms of efficacy and to provide a cost evaluation based on findings of this systematic review. METHODS: The review was conducted selecting prospective, randomized, controlled trials comparing the two gonadotropin medications from a literature search of several databases. The outcome measure used to evaluate efficacy was the number of oocytes retrieved per cycle. In addition, a cost evaluation was performed based on retrieved efficacy data. RESULTS: The number of oocytes retrieved appeared to be higher for human menopausal gonadotropin in only 2 studies while 10 out of 13 studies showed a higher mean number of oocytes retrieved per cycle for recombinant follicle-stimulating hormone. The results of the cost evaluation provided a similar cost per oocyte for both hormones. CONCLUSIONS: Recombinant follicle-stimulating hormone treatment resulted in a higher oocytes yield per cycle than human menopausal gonadotropin at similar cost per oocyte.

Correlation between follicular diameters and flushing versus no flushing on oocyte maturity, fertilization rate and embryo quality.

OBJECTIVE: To determine (a) the correlation between follicular sizes, oocyte maturity, normal fertilization rate, cleavage and embryo quality; and (b) to establish whether oocytes recovered with or without follicular flushing have different developmental competence. DESIGN: Prospective observational study. SETTING: Academic medical center. PATIENTS: Forty nine cycles (37 ICSI and 12 IVF). INTERVENTIONS: Measurement of 360 follicular diameters on the day of egg retrieval and classification into three groups Group A (mean diameter 12-14.5 mm.), group B (mean diameter 15-18 mm.) and group C (diameter >18.5 mm.). MAIN OUTCOME MEASURE: Correlation between follicular size at the time of retrieval and oocyte maturity, fertilization and cleavage rate in 226 oocytes (163 ICSI and 63 IVF). Developmental competence of oocytes retrieved with flushing versus non flushing. RESULTS: Almost all (99 %) of the oocytes recovered from follicles of group C were in metaphase II as opposed to 80 % in group A and 81 % in group B (p < 0.01). Overall there was a progressive and significant increase in fertilization rates from group A follicles to group C (47 % vs. 67 %, p 0.05). Overall 53 % of oocytes retrieved from group A follicles showed either no fertilization or abnormal fertilization versus 27 % in group C (p 0.05). The oocyte recovery rate with follicular flushing improved from group A to group B and to group C follicles (65 % vs. 49 % vs.37 % respectively p < 0.01). There were no differences in rates of immature oocyte, fertilization, abnormal or not fertilization and cleavage. CONCLUSIONS: The results of this study shows that: a) Follicles larger than 18 mm at retrieval have consistently mature oocytes with a higher rate of fertilization; b) Small size follicles are still capable of containing mature oocytes, but their rate of abnormal or no fertilization is high; c) Oocytes recovered with flushing are still able to produce embryos with full developmental competence.

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

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

Preliminary evidence for high anti-PLAC1 antibody levels in infertile patients with repeated unexplained implantation failure.

OBJECTIVE: Placenta-specific1 (PLAC1) is a trophoblast-specific gene encoding for a protein that is highly expressed in human placenta, on the surface of the syncytiotrophoblast. PLAC1 was found to elicit spontaneous antibody responses in cancer patients. We aimed to determine the levels of anti-PLAC1 antibodies in infertile women with a history of unexplained repeated implantation failure after IVF cycles as compared to fertile women. STUDY DESIGN: An observational case-control clinical study. MAIN OUTCOME MEASURE(S): Two groups of patients were analysed in two different experimental settings: 21 infertile women and 81 control patients were enrolled in the first group, 16 infertile women and 67 fertile controls in the second group. Anti-PLAC1 antibody levels and ranking were analysed by ELISA test. RESULTS: In both groups of infertile patients enrolled, optical densities (OD) from ELISA test ranked significantly higher than those of controls (0.27 ± 0.2 vs. 0.13 ± 0.1 respectively; p = 0.0009 in the first group), (0.62 ± 0.38 vs. 0.39 ± 0.35 respectively; p = 0.0044 in the second experiment). In the first group about one case in four (29%) had OD levels above the 95th percentile (0.337) for healthy controls (p = 0.005). In the second experiment 4 out of 16 cases (25%) had OD levels above the 95th percentile (0.878) for healthy controls (p = 0.023). CONCLUSIONS: Anti-PLAC1 antibodies could represent a biomarker associated with infertility and with high probability of repeated implantation failure after ovarian stimulation and IVF-ET, greatly improving the diagnostic work up of infertile couples.

Five years (2004-2009) of a restrictive law-regulating ART in Italy significantly reduced delivery rate: analysis of 10,706 cycles.

STUDY QUESTION: Was the delivery rate of ART cycles negatively affected by the enactment of the Law 40/2004 by the Italian Parliament which imposed a long list of restrictions for ART procedures? SUMMARY ANSWER: This large and extensive comparative analysis of ART outcomes prior to and after the introduction of the Law 40 revealed a significant reduction in pregnancy and delivery rates per cycle, independent of age or other clinical variables, once the law went into effect. WHAT IS KNOWN ALREADY: Several studies have been published on the effect of Law 40/2004 on ART outcomes, some authors demonstrating a negative impact of the Law in relation to specific etiologies of infertility, other authors showing opposite conclusions. STUDY DESIGN, SIZE, DURATION: Retrospective clinical study of 3808 patients treated prior to the enactment of the Law, September 1996-March 2004 (Group I) and 6898 treated during the Law, March 2004-May 2009 (Group II). PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 10 706 ART cycles were analysed, 3808 performed before and 6898 after the application of the Law. An intention-to-treat statistical analysis was performed to detect pregnancy and delivery rates (pregnancies ≥ 24 weeks) per started cycle. A P value of <0.05 was considered statistically significant. We analysed different outcomes: differences in fertilization, pregnancy and delivery rate, multiple pregnancies and miscarriage rates between the two time periods. MAIN RESULTS AND THE ROLE OF CHANCE: The delivery rate for started cycle was 20% before and 16.0% after the introduction of the Law representing a 25% reduction (P < 0.001). The multivariate analysis, corrected by female age of >38 years, duration of infertility, basal FSH level and number of retrieved oocytes, showed a 16% lower delivery rate (odds ratio: 0.84; confidence interval: 0.75-0.94). This statistical approach removed the risk that the observed effects were due to chance and confirmed unequivocally that the Law was an independent factor responsible for the reduced likelihood of a successful outcome. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study. A prospective randomized study, with patients treated in the same time period and randomized to restrictions or not, would have minimized potential limitations due to differences in years of treatments. WIDER IMPLICATIONS OF THE FINDINGS: Our findings based on the analysis of such a large number of cycles proved clearly and unequivocally that imposing restrictions on the practice of ART penalized patients. These data represent a relevant clinical contribution for countries still debating the enactment of restrictive limitations of ART.

Gynecological fertility-sparing surgery.

The implementation of early detection protocols and advanced treatment strategies has significantly improved survival outcomes for gynecologic cancer patients. The improvement of oncological outcomes has led to an increased attention toward Quality of Life issues, including the childbearing potential for young women. Traditionally the surgical treatment of cervical, endometrial and ovarian cancers involves the removal of the uterus and adnexa, irrespective of the impact on fertility and parenthood and regardless of patient desires. For young women affected by gynecological malignancies at an apparently early stage, fertility-sparing procedures could be offered. The aim of our review is to going through the available evidence in the Literature and to evaluate the current state of art regarding fertility-sparing procedures for women with gynecological malignancies in terms of oncological and fertility outcomes.

Italian Constitutional Court modifications of a restrictive assisted reproduction technology law significantly improve pregnancy rate.

BACKGROUND: In May 2009, the Italian Constitutional Court banned most of the limitations of a restrictive law regulating assisted reproduction technology on the grounds that it limited a couple's right to have access to the best possible medical treatment and reduce any possible higher risk of complications. The aim of the study was to compare our results in fresh cycles before and after this change. MATERIALS AND METHODS: We analysed retrospectively 3274 IVF cycles: 2248 before and 1026 after the law was modified. RESULTS: There was no significant difference between the two groups in terms of age, basal FSH levels, years of infertility, the number of previous cycles or the number of oocytes retrieved but the number of oocytes used (2.7 ± 0.6 versus 4.6 ± 1.8; P = <0.001), the number of embryos obtained (2.0 ± 0.9 versus 3.3 ± 1.8; P = <0.001) and transferred (2.2 ± 0.7 versus 2.3 ± 0.7; P = <0.001) were all higher after the removal of the previous restrictions, as was the pregnancy rate per started cycle (23.49% versus 20.42%; P = 0.047). Before modification of the law, the pregnancies were single in 74.11% of the cases (versus 71.43% afterwards), twins in 23.44% (versus 26.89%; P = 0.318) and triplets in 2.46% (versus 1.68%; P = 0.594). CONCLUSIONS: Our preliminary results after the removal of the previous legal restrictions show a higher pregnancy rate per started cycle (3.7% represents a 15% difference) and a positive (albeit non-significant) trend towards a reduction in the number of multiple pregnancies.

Chromosomal abnormalities in miscarriages after different assisted reproduction procedures.

About 10-15% of all recognised pregnancies end in spontaneous abortion and around 60% of these show a chromosomal abnormality. The finding of an abnormal karyotype allows one to avoid unnecessary and controversial testing and treatment, providing accurate reproductive and genetic counselling to the couple. Consequently, chromosome study of products of conception (POC) is routinely performed in our Institute, starting from 2002. Cytogenetic analysis on spontaneous metaphases, obtained using direct method, was successfully performed on a total of 277 cases: 133 from patients who underwent assisted reproductive technology (ART) and 144 samples from infertile couples that had natural conceptions (NC). An abnormal karyotype was observed in 84 (63.2%) cases after ART and in 103 (71.5%) after NC demonstrating that the ART group doesn't seem subjected to a higher cytogenetic risk due to the application of technical procedures and higher than the expected risk in the fertile population. Moreover we did not observe a significant difference in the incidence of chromosome anomalies between intracytoplasmic sperm injection (ICSI) (61.5%) and in vitro fertilisation (IVF) (54.5%). However, in the ICSI with testicular sperm extraction (ICSI-TESE) group, 80% of the cases were abnormal with 50% showing triploid/tetraploid karyotypes. Chromosomal abnormalities were present in 54.5% of miscarriages which occurred after ICSI with cryo-preserved oocytes (cryo-ICSI) and in 85.7% after intra-uterine insemination (IUI). To our knowledge this study analysed the largest number of POCs after ART and NC in an infertile population. Moreover, for the first time the cytogenetic results of POC from different ART procedures were included.

Oocyte cryopreservation.

Over the last few years, there has been renewed interest and scientific debate concerning human oocyte cryopreservation. The aim of this study was to analyse the clinical data coming from our long experience of slow-freezing oocytes. Between 2001 and 2007, 1280 thawing cycles were carried out using oocytes previously frozen by means of a slow 1,2 propaniedol+sucrose protocol. A total of 7585 oocytes were thawed, of which 4409 survived and 3622 were microinjected; 144 clinical pregnancies were obtained. The number of thawing cycles increased from 19 in 2001 to 268 in 2007, and the number of thawed oocytes from 197 to 1652. Although the survival rate was significantly lower in the period 2002-2005 than in the period 2006-2007, pregnancy and implantation rates steadily improved from respectively 6.7% and 2.4% in 2001 to 15% and 8.2% in 2007. Our data demonstrate a clinically important improvement in oocyte crypreservation over the years in a Centres with proved experience, and can be offered as a standard of care not only before cancer treatment but also for couples refusing embryo crypreservation or in countries with very restrictive limitations on embryo or zygote freezing.

Cancer risk in male factor-infertility.

Severe forms of male-factor infertility are associated with an increased risk of testicular cancer and scrotal ultrasonography is widely used for diagnosis. In this study, 2172 male members of infertile couples referred to our Reproductive Medicine Unit were submitted to scrotal ultrasonography and 835 selected patients had been followed during a 2-year period. Eight out of nine neoplastic nodules found at the initial examination were unpalpable and discovered by ultrasonography. Ten tumoral lesions were found in 370 testicular biopsies performed for diagnostic purposes or to extract spermatozoa; and eight additional neoplastic lesions were discovered during the 2-year follow-up of 835 patients. The cumulative rate of neoplastic disease was 3.2%. Thirteen cases (1.5%) were malignant (12 germ cell tumours and one non-Hodgkin lymphoma of testicular origin); the remaining 14 were benign forms (Leydig cell tumours and hyperplasias, Sertoli cell nodules, adenomatoid tumours). Testicular volume (cut-off: 12ml) resulted weakly correlated with germ cell cancer (p=n.s., odds ratio 2.01) while low total sperm count (<40x10(6)) (p=0.002, odds ratio 8.4), previous cryptorchidism (p=0.04, odds ratio 7.5) and hypergonadotrophic hypogonadism (p=0.04, odds ratio 7.9) were associated with an increased risk. But a stronger correlation with germ cell cancer was found in the patients with some utrasonographic anomalies, i.e. testicular microlithiasis (p=0.0015, odds ratio 37.1) or larger calcifications not fitting the description of testicular microlithiasis (p<0.0001, odds ratio 69.5). Our findings indicate that scrotum ultrasonography should always be advised in subfertile men with <40x10(6) spermatozoa/ejaculate or hypergonadotrophic hypogonadism or previous cryptorchidism, and that particular care should be taken in the presence of testicular microlithiasis or testicular calcifications. These men should be aware of the existence of higher risk of testicular cancer and trained in testicular self-examination.

Clinical and molecular cytogenetic studies in three infertile patients with mosaic rearranged Y chromosomes.

Isodicentrics (idic) are structural anomalies of the Y chromosome associated with a 45,X cell line and a broad spectrum of phenotypes. We characterized the rearranged Y chromosomes from three azoospermic males by fluorescence in-situ hybridization (FISH) and PCR. Chromosome study was performed on lymphocytes and testicular biopsy. FISH analysis and PCR established the degree of mosaicism and analysed specific Y regions. Two patients showed a 45,X/46,X,?idic(Y) karyotype with varying degrees of mosaicism. FISH demonstrated the presence of two centromeres and two SRY regions. In the lymphocytes of the third patient, the presence of a small Y-derived marker was also observed. An additional cell line with two idic(Y) was present in the testicular biopsy of the same patient. PCR showed the breakpoint between SY182 (KALY) and SY121 in Yq11.221-q11.222 region in all the cases. For the evaluation of the mosaicism, different tissues must be investigated. The phenotypical sex depends more on the number of copies of the SRY gene rather than on the percentage of 45,X cells, at least in the gonads. The combined use of classical and molecular cytogenetics is necessary for delineating the chromosome regions involved allowing a better genotype-phenotype correlation.