Elena Albani

The Impact of Embryo Transfer on Implantation—A Review

Embryo transfer has received little clinical attention and has been, until recently, the most inefficient step in in-vitro fertilization (IVF). In this article, the authors review the literature and their personal experience regarding the process of intrauterine transfer of embryos, which remains the object of much discussion. Factors which appear to influence implantation rates are: contamination of the catheter tip with cervical bacteria, stimulation of uterine contractions during the procedure, the type of catheter, ultrasound guidance during the transfer, and the position of the embryos in the uterine cavity. Easy and atraumatic transfer is essential for successful implantation and the embryos need to be placed in the middle of the cavity, away from the fundus. Knowing, beforehand, the position and length of the uterus can provide better results and may reduce the rate of ectopic pregnancies. Evidence from randomized studies has supported this claim. Despite the number of available studies controlling certain variables, most authors, even using the same catheter, ultrasound guidance and/or a trial transfer use different protocols or similar instruments in different ways. Standardization of the transcervical intrauterine transfer of embryos in a large randomized study is needed before definitive conclusions can be drawn. The goal of improved implantation and pregnancy rates deserve these efforts.

Sperm protein 17 is expressed in the sperm fibrous sheath

BackgroundSperm protein 17 (Sp17) is a highly conserved mammalian protein characterized in rabbit, mouse, monkey, baboon, macaque, human testis and spermatozoa. mRNA encoding Sp17 has been detected in a range of murine and human somatic tissues. It was also recognized in two myeloma cell lines and in neoplastic cells from patients with multiple myeloma and ovarian carcinoma. These data all indicate that Sp17 is widely distributed in humans, expressed not only in germinal cells and in a variety of somatic tissues, but also in neoplastic cells of unrelated origin.MethodsSp17 expression was analyzed by immunocytochemistry and transmission electron microscopy on spermatozoa.ResultsHere, we demonstrate the ultrastructural localization of human Sp17 throughout the spermatozoa flagellar fibrous sheath, and its presence in spermatozoa during in vitro states from their ejaculation to the oocyte fertilization.ConclusionThese findings suggest a possible role of Sp17 in regulating sperm maturation, capacitation, acrosomal reaction and interactions with the oocyte zona pellucida during the fertilization process. Further, the high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP)-binding motif within this region, suggest that Sp17 might play a regulatory role in a protein kinase A-independent AKAP complex in both germinal and somatic cells.

Implantation failure in assisted reproduction technology and a critical approach to treatment.

Abstract: In this article, we review the literature and our personal experience regarding the many factors that appear to influence implantation rate. Oocyte quality, as determined by patient age and aneuploidies, probably plays a major role in RIF. However, a panoply of other factors have been brought under investigation, quite often with contradictory results and additional intriguing questions to be studied. Infections of the vagina, cervix, and endometrium, the role of mucus aspiration and washing of the cervix on transfer, the role of catheter guidance for a correct transfer and potion of embryos, the effect of mock transfer, and the role of hysteroscopy and its timing before transfer procedures are analyzed both as a review of the literature and as opinions and data from our experience. Many of these factors are interlaced and from the apparently simple issue of trauma, to infections and immune modulation of hatching and implantation, a biological continuum can easily be identified. The impact of abnormalities of the immune system and of homeostasis abnormalities is also covered in a brief overview of reported works and our experience. These latter areas probably constitute the common biological background of all other external factors that, however, the skilled must equip themselves for improving implantation success.

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 couples 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.

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.

Experimental contamination assessment of a novel closed ultravitrification device

OBJECTIVE To evaluate the safety of a new ultravitrification closed device. DESIGN Ultravitrification research. SETTING Private assisted reproduction center. ANIMAL(S) Microorganisms (bacteria). INTERVENTION(S) A styrofoam container holding 1,000 mL of liquid nitrogen (LN2) was contaminated with Pseudomonas aeruginosa and Escherichia coli. Forty closed devices (Ultravit) and 20 open devices (Cryotop) loading approximately 0.5 μL of antibiotic-free medium were plunged into this contaminated LN2 for 5-10 seconds and then inoculated into selective culture dishes. Colony-forming units were analyzed and counted after an overnight incubation at 37°C. MAIN OUTCOME MEASURE(S) Detection of micro-organisms in different devices after incubation. RESULT(S) There was no contamination in any of the closed devices, whereas in 45% of open devices these bacteria were present. CONCLUSION(S) With this study we demonstrated, in an experimental model using contaminated LN2, that this new closed device is a safe system that does not allow cell contact with LN2, avoiding cell contamination.

Comparative analysis of fetal and neonatal outcomes of pregnancies from fresh and cryopreserved/thawed oocytes in the same group of patients

OBJECTIVE To analyze the fetal and neonatal outcomes of pregnancies achieved with fresh and/or frozen oocytes in the same group of patients. DESIGN Observational study and comparative analysis. SETTING Research unit of an academic medical center. PATIENT(S) A group of 855 women with cryopreserved oocytes and their resulting 954 assisted reproductive technology clinical pregnancies were enrolled and followed up during the same time period and in the same clinical setting; the outcomes of 197 pregnancies from frozen/thawed oocytes were compared with 757 obtained from fresh sibling oocyte cycles. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Pregnancies were followed until delivery, and neonatal data (up to 28 days after delivery) were collected. RESULT(S) No significant differences were found between the use of fresh and frozen oocytes in the rates of therapeutic abortions for fetal anomaly (1.5% vs. 0.8%) and ectopic pregnancies (3.6% vs. 2.9%), but a significantly higher rate of spontaneous abortions at ≤ 12 weeks (17.6% vs. 26.9%) was observed in the frozen/thawed oocytes group. No statistical differences were found in major anomalies at birth (2.8% vs. 4.6%). Despite no difference in gestational age at delivery, the mean birth weights were significantly lower with fresh oocyte pregnancies, both in singleton (2,725 ± 727 g) and twins (2,128 ± 555 g), than with frozen-thawed oocytes (3,231 ± 615 g and 2,418 ± 492 g, respectively). However, the analysis of the 63 patients who obtained pregnancies both in fresh and thawed cycles (138 pregnancies) showed no differences in the abortion rate and in the mean birth weight. CONCLUSION(S) These results provide strong support to the notion that fetal and perinatal complications and congenital anomalies do not differ between pregnancies from frozen-thawed and fresh oocytes. The significantly lower mean birth weight observed with pregnancies from fresh oocytes supports similar observations reported for pregnancies from embryo cryopreservation and requires further prospective studies.

Human oocyte ultravitrification with a low concentration of cryoprotectants by ultrafast cooling: a new protocol

OBJECTIVE To evaluate the efficacy of a new ultravitrification technique with a low concentration of cryoprotectants. DESIGN Ultravitrification research. SETTING Private assisted reproduction center. PATIENT(S) Oocytes donated voluntarily with the aim of research. INTERVENTION(S) Ultravitrification with different protocols of 100 mature oocytes and 100 immature oocytes divided in four groups to determine which is the adequate cryoprotectant concentration and the appropriate cooling solution. MAIN OUTCOME MEASURE(S) Human oocytes survival rate with low concentration of cryoprotectants by ultravitrification technique. RESULT(S) We obtained higher survival rates with slush nitrogen than with liquid nitrogen (92% vs. 56%) and better results with 2 M of cryoprotectants than with 1.5 M (92% vs. 60%). The best protocol was 2 M PrOH + 0.5 M sucrose + slush nitrogen with a mature oocytes survival rate of 92% (23 of 25) and immature of 88% (22 of 25). CONCLUSION(S) This ultravitrification technique is a new option to preserve human oocytes that avoids the use of a high cryoprotectant concentration while obtaining a high survival rate with a concentration of cryoprotectants typical of slow freezing.

Ejaculate Oxidative Stress Is Related with Sperm DNA Fragmentation and Round Cells

Oxidative stress (OS) plays an essential role in male infertility aetiology by affecting sperm quality, function, and also the integrity of sperm DNA. The assessment of oxidative stress in semen may be an important tool to improve the evaluation of sperm reproductive capacity. The purpose of this study was the evaluation of any possible relation between the unbalance of oxidative stress caused by superoxide anion in the ejaculate with the presence of sperm DNA fragmentation and high concentration of round cells. 56 semen samples from males from couples suffering from infertility were evaluated according to World Health Organisation (WHO) 2010 guidelines. Oxidative stress levels from N1 (low) to N4 (high) were assessed in ejaculates using oxiSperm; DFI (sperm DNA fragmentation index) as assessed by the SCSA (Sperm Chromatin Structure Assay) was used for evaluation of sperm chromatin integrity. Our data show that high oxidative stress (N3-N4 levels) correlated positively with a DFI ≥ 30% (P = 0.0379) and round cells ≥1.500.000/mL (P = 0.0084). In conclusion, OS increases sperm DNA damage. Thus evaluation of semen OS extent of sperm DNA damage in infertile man could be useful to develop new therapeutic strategies and improve success of assisted reproduction techniques (ART).

ICSI outcome is significantly better with testicular spermatozoa in patients with necrozoospermia: a retrospective study

Abstract Objective: To determine whether intracytoplasmic sperm injection (ICSI) outcome with testicular sperm is superior to that of ejaculated sperm in men with incomplete necrozoospermia, defined as sperm viability ≥5 to ≤45%. Study design: Retrospective study at a Reproductive Medicine Center of a tertiary referral hospital. A total of 231 couples with male infertility due to incomplete necrozoospermia underwent 342 ICSI cycles with freshly ejaculated sperm (ICSI-ejaculated) and 182 cycles with testicular sperm (ICSI-TESE). Results: Overall 1624 MII oocytes were injected in the ICSI-ejaculated group with a fertilisation rate of 60.8%, while in ICSI- TESE cycles the fertilisation rate was 59.6% in 874 MII oocytes. The cleavage rate was higher in ICSI-ejaculated cycles than in ICSI-TESE cycles (96.3% versus 92.9%; p = 0.004). However, the pregnancy and implantation rates per cycle were significantly higher in the ICSI-TESE group (67/182, 36.8% versus 68/342, 19.9% (p = 0.0001); and 23.7% versus 12.7% (p = 0.0001), respectively). The miscarriage rate was similar (ICSI-ejaculated 26.5% versus ICSI-TESE 17.9%, p = 0.301). Live birth rate per cycle in the ICSI-ejaculated group was significantly lower than in the ICSI-TESE (13.7% versus 28.6%, p = 0.0001). Conclusions: In cases of persistent necrozoospermia, testicular sperm should be favoured over ejaculated sperm. These data call for more research to understand the pathophysiology of refractory necrozoospermia.