Cumulus cells surrounding oocytes with high developmental competence exhibit down-regulation of phosphoinositol 1,3 kinase/protein kinase B (PI3K/AKT) signalling genes involved in proliferation and survival.

STUDY QUESTION: Is the phosphoinositol 1,3-kinase/protein kinase B (PI3K/AKT) pathway expression profile in cumulus cells (CCs) a potential marker of oocyte competence and predictive of pregnancy outcome? SUMMARY ANSWER: Eleven genes (AKT1, ARHGEF7, BCL2L1, CCND1, E2F1, HRAS, KCNH2, PIK3C2A, SHC1, SOS1 and SPP1) in the PI3K/AKT pathway were significantly down-regulated in CCs from oocytes that went on to produce a pregnancy compared to CCs associated with a negative outcome. WHAT IS KNOWN ALREADY: The PI3K/AKT pathway plays a pivotal role in the interdependence and continuous feedback between the oocyte and CCs. STUDY DESIGN SIZE, DURATION: The expression analysis of 92 transcripts in the PI3K/AKT pathway in CCs from patients with negative or positive pregnancy outcome, after single embryo transfer, was performed. Mouse CCs target gene expression was conducted to associate the expression profile of PI3K/AKT pathway to oocyte developmental profile. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fifty-five good prognosis IVF patients who had been referred to IVF or intracytoplasmic sperm injection treatment for male-factor infertility or tubal disease were enroled. CCs from single cumulus-oocyte complexes (COCs) from 16 patients who underwent a single embryo transfer were analyzed. Twenty-five CD-1 mice were used to assess gene expression in CCs associated with oocytes with different competence in relation to hCG priming. A total 220 human COCs were collected. The RNA extracted from CCs of 16 selected patients was used to analyze PI3K/AKT pathway gene expression employing a 96-well custom TaqMan Array. Expression data of CCs associated to positive IVF outcome were compared to data from negative outcome samples. Mice were sacrificed after 9, 12, 15, 21 and 24 h post-hCG administration to obtain CCs from MII oocytes with different developmental competence. Akt1, Bcl2l2 and Shc1 expression were tested in the collected mouse CCs. In addition, the expression of upstream regulator ESR1, the gene encoding for the oestrogen receptor ERß, and the downstream effectors of the pathway FOXO1, FOXO3 and FOXO4 was evaluated in human and mouse samples. MAIN RESULTS AND THE ROLE OF CHANCE: Transcripts involved in the PI3K Signaling Pathway were selectively modulated according to the IVF/ICSI outcome of the oocyte. Eleven transcripts in this pathway were significantly down-regulated in all samples of CCs from oocytes with positive when compared those with a negative outcome. These outcomes were confirmed in mouse CCs associated with oocytes at different maturation stages. Expression data revealed that the down-regulation of ESR1 could be related to oocyte competence and is likely to be the driver of expression changes highlighted in the PI3K/AKT pathway. LIMITATIONS REASONS FOR CAUTION: Small sample size and retrospective design. WIDER IMPLICATIONS OF THE FINDINGS: The CCs expression profile of PI3K/AKT signaling genes, disclosed a specific CCs gene signature related to oocyte competence. It could be speculated that CCs associated with competent oocytes have completed their role in sustaining oocyte development and are influencing their fate in response to metabolic and hormonal changes by de-activating anti-apoptotic signals. STUDY FUNDING/COMPETING INTEREST(S): Supported by Merck Serono an affiliate of Merck KGaA, Darmstadt, Germany (research grant for the laboratory session; Merck KGaA reviewed the manuscript for medical accuracy only before journal submission. The authors are fully responsible for the content of this manuscript, and the views and opinions described in the publication reflect solely those of the authors). The authors declare no conflict of interest.

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

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

Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage.

BACKGROUND: Sperm DNA integrity is essential for the accurate transmission of genetic information. The clinical significance of this assessment lies in its association with not only natural conception rates, but also the success of assisted reproduction technology (ART). It has been reported that sperm chromatin structure assay (SCSA) identified thresholds for negative pregnancy outcome after ART when the DNA fragmentation index (DFI), previously known as COMPalphat, was >30%. METHODS: In a prospective clinical study, we examined 34 male infertile patients, the husbands of women undergoing conventional IVF or ICSI. SCSA and ART were carried out on semen aliquots taken from the same ejaculate. Fertilization rate, embryo quality and pregnancy rates were correlated to SCSA parameters, DFI and highly DNA stainable (HDS) cells. RESULTS: No differences were seen in SCSA parameter values between patients initiating pregnancies and not doing so in either ICSI or conventional IVF. Pregnancies and normal delivery were obtained even with high levels of DFI. CONCLUSIONS: There is still controversy over whether analytical techniques currently in use are able to identify the level of damage to spermatozoa. Large-scale studies should be conducted in different clinical settings to determine the effects of sperm DNA damage on the outcome of ART.

Putative hepatotoxic neoclerodane diterpenoids from Teucrium species.

Fourteen neo-clerodane diterpenoids have been isolated from the acetone extract of the aerial parts of four species of Teucrium (T.alpestre, T. cuneifolium, T. divarication subsp. villosum, and T. flavum subsp. hellenicum) not previously studied chemically.

Vero cell effect on in-vitro human blastocyst development: preliminary results.

Asynchrony between embryo and uterine environment is one of the major limits in human in-vitro fertilization (IVF). A culture system which could prolong culture time and increase embryonic cleavage rate and viability would improve success rates. Using Vero cells, an in-vitro co-culture system was developed to investigate and promote human embryo development. Vero cells provide good support for human early embryos up to the blastocyst stage. When fertilized embryos were co-cultured, 68% of them reached the blastocyst stage. Pregnancy rate was 50% per transfer in patients with several previous failures of implantation. A significant increase in clinical pregnancy rate was also demonstrated when zygotes were maintained on Vero cell monolayer for only 24 h. The beneficial effect of the feeder layer may be through the release of embryotrophic factors and the detoxification of the culture medium by the cells. Co-culture is a new concept in assisted reproduction.

What is the most idoneous developmental stage for embryo-freezing?

Mammalian preimplantation embryos can be cryopreserved by a variety of methods involving different cryoprotectants. In the present study, we ultrarapidly froze 299 excess embryos at various early cleavage stages (zygotes, 2-cell embryos, 4-cell embryos and > 5-cell ones) after a brief exposure to high concentrations of dimethyl sulfoxide (DMSO; 3.5 M) and 0.25M sucrose. One-hundred seventeen of them were thawed in a warm water bath. Thirtyone embryos were completely destroyed after thawing but only 6% of them were originally at the pronuclear stage. Regarding the morphological aspects, 94% of pronucleate embryos were intact whereas just 12% of > 5-cell embryos showed more than 50% intact blastomeres after thawing. We froze also 60 embryos at the blastocyst stage by a slow freezing procedure. Eighteen blastocysts were thawed and 12 of them fully re-expanded were transferred in four patients.

Isolation of a histamine releasing factor from human embryo culture medium after in-vitro fertilization.

In-vitro fertilization (IVF) of human oocytes in our laboratories gave a percentage pregnancy rate per transfer close to 20% during 1985. Embryos were grown until the two-four cell stage and then transferred to the maternal uterus. The media from these embryo cultures were collected and subjected to chromatography on heparin-Sepharose affinity columns. The bound protein fraction contained a factor capable of inducing histamine release from sensitized basophils. The effect of this embryo-derived histamine-releasing factor (EHRF) was to induce a maximum 56 +/- 7% release of the total histamine available. This value varied between 20 and 60%, resulting from 10-30 micrograms/ml of EHRF. Since the histamine release assay performed with basophils from non-atopic donors gave no positive results, we conclude that the release was not due to a cytotoxic mechanism. This was also supported by the absence of histamine release when the assay was performed at 0 degree C, or in the presence of 2 mM EDTA, suggesting that release was dependent on an immunological interaction between EHRF and some receptor on the basophils. The immunosuppressive role of histamine is well known, and a model involving EHRF and histamine is suggested here to explain the mechanism mounted by the embryo to escape maternal immune rejection.

Gamete intrafallopian transfer in the treatment of infertility: the first series at the University of Palermo.

Twenty-six couples with unexplained infertility (UI), nine women with repeated failures of artificial insemination with donor semen (AID), three women with mild endometriosis, three with periadnexal adhesions, one with hostile (not immunologic) cervical mucus, and one couple in which the male partner was affected by asthenospermia were treated by the gamete intrafallopian transfer (GIFT) technique. Three different protocols for controlled ovarian hyperstimulation were used, and an adequate follicular growth and oocyte maturation were achieved in all cases. Seventeen pregnancies were obtained, for a global pregnancy rate of 38.6%. Two pregnancies (11.7%) ended in clinical abortions, and one (5.8%) was a tubal pregnancy. Of the ongoing pregnancies, one is twin and two are triplets. Seven pregnancies (six ongoing, one abortion) were obtained in the UI group (26%), six (all ongoing) in the failed AID group (66.6%), two continuing pregnancies in the three patients with endometriosis (66.6%); the tubal pregnancy and one clinical abortion occurred in the group with adnexal adhesions. No pregnancies were obtained in the patient with hostile cervical mucus and in the couple with infertility presumably due to poor semen. These encouraging results and the simplicity of the technique suggest that GIFT could be an effective approach that could be programmed during a well-timed laparoscopy where persistent infertility exists in association with apparently normal fallopian tubes.

One year's experience with the GIFT method.

From January 1985 to December 1985 we have performed 83 GIFTS for various indications such as unexplained infertility, immunological problems, endometriosis, male factor, fimbrial pathology. In 82 women out of 83, oocytes and sperm have been transferred and 31 pregnancies (37.8%) have been obtained. Twelve women have delivered 17 babies; 11 pregnancies are ongoing, 7 have been interrupted by an abortion and 1 by a tubal pregnancy. The present and future roles of GIFT in the treatment of infertility is discussed.

Gamete intra-fallopian transfer (GIFT): a new technique for the treatment of unexplained infertility.

The Gamete Intrafallopian Transfer (GIFT) was carried out in 12 couples with unexplained infertility, in 2 cases with infertility associated with mild endometriosis, in 1 case of hostile mucus and 3 cases in which phymosis of the ampulla and/or periadnexal adhesions were previously identified. In 7 couples GIFT was carried out after several AID cycles for husband azoospermia or severe oligospermia. Three different protocols were used for the controlled ovarian hyperstimulation. In all cases an adequate follicular growth was obtained and mature oocytes were recovered. At first attempt 12 ongoing pregnancies (1 triplet, 1 twin) were obtained with a present pregnancy rate of 48%.