Rubens Fadini

Cleavage kinetics analysis of human embryos predicts development to blastocyst and implantation

Cleavage kinetics of human embryos is indicative of ability to develop to blastocyst and implant. Recent advances in time-lapse microscopy have opened new and important research opportunities. In this study involving infertile couples requiring standard IVF/intracytoplasmic sperm injection treatment, zygotes were cultured by integrated embryo-culture time-lapse microscopy to analyse cleavage times from the 2- to the 8-cell stages in relation to the ability to develop to blastocyst, expand and implant. In comparison to embryos arresting after 8-cell stage, times of cleavage to 7- and 8-cell stages of embryos developing to blastocyst were shorter (56.5 ± 8.1 versus 58.8 ± 10.4h, P=0.03 and 61.0 ± 9.4 versus 65.2 ± 13.0 h, P=0.0008, respectively). In embryos developing to blastocyst, absence of blastocoele expansion on day 5 was associated with progressive cleavage delay. Implanting embryos developed to 8-cell stage in a shorter period compared with those unable to implant (54.9 ± 5.2 and 58.0 ± 7.2h, respectively, P=0.035). In conclusion, cleavage from 2- to 8-cell stage occurs progressively earlier in embryos with the ability to develop to blastocyst, expand and implant. Conventional observation times on days 2 and 3 are inappropriate for accurate embryo evaluation. The speed at which human embryos cleave is known to be suggestive of their ability to develop in vitro to the blastocyst stage and implant after transfer into the uterus. Recent advances in time-lapse microscopy, which allows acquisition of images every 15-20 min, have opened new and important research opportunities. In a retrospective study involving infertile couples requiring standard IVF or intracytoplasmic sperm injection treatment, fertilized oocytes were cultured by an integrated embryo-culture time-lapse microscopy system in order to perform an analysis of cleavage times from the 2- to the 8-cell stage in relation to the ability to develop to blastocyst, expand and implant. In comparison to embryos arresting after the 8-cell stage, times of cleavage to the 7- and 8-cell stage of embryos that developed to blastocyst were significantly shorter (56.5 ± 8.1h versus 58.8 ± 10.4h and 61.0 ± 9.4h versus 65.2 ± 13.0 h, respectively). In embryos developing to the blastocyst stage, absence of blastocoele expansion on day 5 was associated with a progressive cleavage delay. Implanting embryos developed to the 8-cell stage in a shorter period compared to those unable to implant (54.9 ± 5.2h and 58.0 ± 7.2h, respectively, P=0.035). In conclusion, cleavage from the 2- to the 8-cell stage occurs progressively earlier in embryos with the ability to develop to blastocyst, expand and implant. Conventional observation times on day 2 and 3 are appropriate for accurate embryo evaluation.

Human oocyte maturation in vitro

Oocytes from medium-sized antral follicles have already completed their growth phase and, if released from the follicular environment and cultured in vitro, are able to resume the meiotic process and mature. However, in vitro maturation (IVM) does not entirely support all the nuclear and cytoplasmic changes that occur physiologically as an effect of the ovulatory stimulus. Regardless, oocyte IVM is widely applied for the breeding of agriculturally important species. In assisted reproduction technology, IVM has been proposed as an alternative treatment to circumvent the drawbacks of standard ovarian stimulation regimens. Initially introduced to eliminate the risks of ovarian hyperstimulation syndrome afflicting women presenting with polycystic ovaries, subsequently IVM has been suggested to represent an additional approach suitable also for normovulatory patients. So far, in children born from IVM cycles, no doubts of an increased incidence of congenital abnormalities have been raised. Many more births would be achieved if novel IVM systems, currently dominated by empiricism, could be conceived according to more physiological criteria. Recent findings shedding new light on the control of meiotic progression, the support of cumulus cells to the oocyte cellular reorganization occurring during maturation, and the modulation of the stimulus that promotes oocyte maturation downstream the mid-cycle gonadotropin signal are likely to provide crucial hints for the development of more efficient IVM systems.

Zoladex (goserelin acetate) and the anemic patient: results of a multicenter fibroid study * † ‡ § ∥

OBJECTIVE To compare the effects of goserelin acetate treatment with or without iron with iron alone. DESIGN Multinational, multicenter, prospective, randomized, double-blind study. PATIENTS Premenopausal women with menorrhagia or metrorrhagia and anemia associated with uterine leiomyomata awaiting hysterectomy. INTERVENTION Patients were randomized to one of three 12-week treatment groups namely goserelin acetate 3.6 mg once monthly plus placebo iron; 3.6 mg goserelin acetate once monthly plus 600 mg/d iron; or sham injection once monthly plus 600 mg/d iron. MAIN OUTCOME MEASURE Preoperative hemoglobin concentration; preoperative uterine and fibroid volumes and operative blood loss. RESULTS Considering the entry and preoperative hemoglobin concentrations, there was a difference in least square means of just over 1 g/dL between the goserelin acetate plus iron and iron only groups and 2.6 g/dL between the goserelin acetate plus iron and goserelin acetate only group. These differences were both statistically significant. Uterine and fibroid volumes were decreased in the goserelin acetate-treated patients by between 37% and 40% and 44% and 47%, respectively, compared with 7% decreases for both in the iron only group. The differences in absolute changes were statistically significant for both the goserelin acetate-treated groups versus the iron-treated group. The least square geometric mean operative blood loss was greatest in the iron only group. CONCLUSION In the patient with uterine leiomyomata and anemia, goserelin acetate in combination with iron therapy has shown significant advantages over the iron alone in restoring hematologic normality, decreasing uterine and fibroid volumes, and reducing operative blood loss.

The current challenges to efficient immature oocyte cryopreservation

Oocyte cryopreservation represents an important tool for assisted reproductive technology. It offers the opportunity to preserve fertility in women at risk of loss of the ovarian function for various pathologies. It also represents a treatment alternative for couples that cannot benefit from embryo cryopreservation because of moral, religious, or legal constrains. On the other hand, in vitro oocyte maturation has a range of applications. It can be applied in patients with a contraindication to ovarian stimulation to prevent ovarian hyperstimulation syndrome or to eliminate the risk of stimulation of hormone-sensitive tumours in cancer patients. However, while mature oocyte cryopreservation has found wide-spread application and oocyte in vitro maturation has a place for the treatment of specific clinical conditions, data on the efficiency of freezing of immature or in vitro matured oocytes are poorer. In this review we will focus on the combination of oocyte in vitro maturation with oocyte cryopreservation with particular emphasis on the biological implications of the cryopreservation of immature or in vitro matured oocytes. The two cryopreservation approaches, slow freezing and vitrification, will be discussed in relation to possible cryodamage occurring to subcellular structures of the oocyte and the functional interaction between oocyte and cumulus cells.

Cumulus cell-oocyte complexes retrieved from antral follicles in IVM cycles: relationship between COCs morphology, gonadotropin priming and clinical outcome.

PurposeTo assess retrospectively the developmental potential of different types of cumulus cell-oocyte complexes (COCs) derived from IVM cycles.MethodsIVM cycles were performed in natural cycles or after HCG, FSH, or FSH/HCG priming. COCs recovered were morphologically characterized in different types: compact (CC) or expanded (EC) cumulus mass but including an immature oocyte, and expanded cumulus mass enclosing a mature oocyte (EC-MII). Embryo developmental competence was investigated analysing exclusively cycles in which all transferred embryos derived from the same COC category.ResultsFertilization rates did not differ significantly. Significant differences in pregnancy rates (14.5 %, 10.0 % and 27.6 % in the CC, EC, and EC-MII categories, respectively) were observed. Likewise, significant differences in implantation rates (8.9 %, 6.3 % and 19.1 % in the CC, EC, and EC-MII categories, respectively) were found. Overall, priming with FSH/HCG had a beneficial effect on pregnancy and implantation rates, while no priming or HCG alone generated oocytes with poor competence.ConclusionsIn IVM cycles, morphological evaluation at the time of collection can predict the developmental ability of different COCs. FSH/HGC priming has a positive effect on oocyte competence.

Embryo transfer following in vitro maturation and cryopreservation of oocytes recovered from antral follicles during conservative surgery for ovarian cancer.

IntroductionTherapeutic advances have significantly improved the prog-nosis of cancer patients [1]. This has generated new expect-ations especially for women of adolescent and reproductiveage for whom an increased hope of recovery from diseaseimplies a prospect of parenthood [2]. Unfortunately, radio-and chemotherapies have major effects on ovarian function,often leading to premature ovarian failure. Over the lastseveral years, different strategies have been developed topreserve female germ cells and, with them, reproductivefunction. Before a cancer treatment is started, parts of ovar-ian cortex can be explanted, cryopreserved and re-implantedorthotopically after clinical remission. With this approach,ovarian and reproductive function can be restored, at leasttransiently, as demonstrated by the birth of naturally con-ceived children [3]. Alternatively, following controlledovarian stimulation, mature oocytes can be retrieved, cryo-preserved and used at later stages to achieve a pregnancywith embryos generated in vitro [4]. Retrieval and storage ofimmature or in vitro matured oocytes may offer an addition-al opportunity for female germ cell preservation in cancerpatients [5]. In fact, germinal vesicle (GV)-stage oocytescan be collected from antral follicles in the absence ofgonadotropin administration and cryopreserved before orafter in vitro maturation (IVM). Therefore, IVM may bepreferable in cases in which tumour estrogen-sensitivityand/or urgency to start therapy conflict with the implemen-tation of a controlled ovarian stimulation treatment. In thisreport, we describe the recovery of immature oocytes fromantral follicles during a laparotomic conservative surgery forovarian cancer. These oocytes were matured in vitro, cry-opreserved by vitrification at the mature stage and subse-quently warmed to pursue a pregnancy in an IVF cycle. Thisexperience offers the proof of principle that opportunisticretrieval of immature oocytes during surgery is a realisticpossibility to preserve female reproductive potential.Case reportIn July 2010, a 38-year-old woman with a previous historyof infertility (one spontaneous abortion in 2005, three intra-uterine inseminations in 2008, and one ICSI cycle in April2010) underwent laparotomic surgery for ovarian adenocar-cinoma. The tumour displayed moderate differentiation(G2) and was staged as IIC according to the FIGO classifi-cation [6]. In the course of intervention, the extent andseverity of lesions required contextual right annexectomyand excision of an intact cyst from the left ovary.In consideration of the clinical condition and desire ofparenthood by the patient, and with approval of the local

Contributions of the actin cytoskeleton to the emergence of polarity during maturation in human oocytes

In mature mammalian oocytes, cortical f-actin distribution is polarized, as evidenced by a prominent cap subtended by the metaphase II (MII) spindle. Formation of a polarized actin cap is a consequence of a complex actomyosin-driven contractile process that directs polar body extrusion. Human mature oocytes also display a network of suboolemmal actin, but so far there has been no suggestion of an actin-rich domain in the vicinity of the spindle. By high-resolution confocal microscopy, we generated semi-quantitative data of the actin cytoskeleton in human mature and immature oocytes, with the aim to better understand the characteristics and remodelling of this cytoskeletal component in the female gamete. In mature MII oocytes, the cortical domain near the spindle showed a more intense actin signal in comparison to the opposite cortical domain (177.2±59.0 versus 126.8±61.0, P<0.0001; data expressed in arbitrary units). The extent of cortical f-actin polarity was comparable between in vivo and in vitro matured oocytes. However, both the degree of polarity and relative abundance of signal were diminished with increasing maternal age. Mean intensity of cytoplasmic actin was significantly higher in oocytes matured in vitro derived from in vitro maturation (IVM) cycle, in comparison to oocytes matured in vivo or in vitro obtained from controlled ovarian stimulation cycles (35.0±8.0, 21.1±12.4 and 25.9±8.6, respectively; P=0.025). In germinal vesicle (GV)-stage oocytes obtained from both IVM and controlled ovarian stimulation cycles, cortical actin did not appear polarized, irrespective of whether the GV was located centrally or asymmetrically. These data indicate that, during maturation, cortical actin acquires a polarized distribution involving an accumulation in the domain adjacent the spindle. They also propose new questions concerning the existence of cytoplasmic actin in mature oocytes. Finally, they are suggestive of an influence of maternal age on the actin cytoskeleton.

Expression and regulation of sFRP family members in human granulosa cells

Follicular development and ovulation are major processes in the reproductive system. Understanding their complexity is important to female fertility treatments and the control of reproductive processes. Wnt signaling pathway components were shown to be involved in reproduction in animal models. The secreted frizzled-related protein-4 (sFRP4), a potential modulator of Wnt4 signaling pathway, was shown to be induced by LH in rodents and expressed in the corpus lutea, but the pattern of its expression in human ovaries remains unknown. We evaluated the expression pattern of sFRP4 and other sFRP family members in human mural and cumulus granulosa cells (GCs), as well as their regulation by LH/hCG. GCs were obtained from follicles aspirated during in vitro maturation and IVF procedures. GCs were also plated and grown in culture. We showed that the human sFRP4 expression decreases as follicles grows to the preovulatory stage and its expression was higher in cumulus GCs than in mural GCs. Interestingly, LH/hCG stimulation of GCs in vivo and in culture resulted in decreased expression of sFRP4. Of the other sFRP family members, sFRP5 expression was found in mural and cumulus GC in vivo and was shown to be induced by LH/hCG in vitro and in vivo. In summary, sFRP4 is expressed in human GCs and its expression declines during late antral follicular growth. sFRP4 expression is also inhibited by LH/hCG, unlike its rodent homolog. In human GC, sFRP5 may substitute the role of sFRP4 in mouse GC.

Clomiphene citrate versus high doses of gonadotropins for in vitro fertilisation in women with compromised ovarian reserve: a randomised controlled non-inferiority trial

BackgroundThe aim of the present randomised controlled non-inferiority trial is to test whether in women with compromised ovarian reserve requiring in vitro fertilisation, a protocol of ovarian stimulation using exclusively clomiphene citrate performs similarly to a regimen with high doses of gonadotropins.MethodsWomen with day 3 serum FSH > 12 IU/ml on at least two occasions or previous poor response to hyper-stimulation were recruited at four Italian infertility units. Selected women were allocated to clomiphene citrate 150 mg/day from day 3 to day 7 of the cycle (n=145) or to a short protocol with GnRH agonist 0.1 mg and recombinant FSH 450 IU daily (n=146). They were randomised by means of a computer-generated list into two groups. The study was not blinded. The main outcome of the study was the delivery rate per started cycle.ResultsThe study was interrupted after the scheduled two years of recruitment before reaching the sample size. 148 women were allocated to clomiphene citrate and 156 to the short protocol with high doses of gonadotropins; 124 and 125 participants were analysed in the groups, respectively. Women allocated to high doses of gonadotropins retrieved more oocytes and had a higher probability to perform embryo-transfer. However, the chances of success were similar. The delivery rate per started cycle in women receiving clomiphene citrate and high-dose gonadotropins was 3% (n=5) and 5% (n=7), respectively (p=0.77). The mean estimated cost per delivery in the two groups was 81,294 and 113,107 Euros, respectively. No side-effects or adverse events were observed.ConclusionsIn women with compromised ovarian reserve selected for in vitro fertilisation, ovarian stimulation with clomiphene citrate or high-dose gonadotropins led to similar chances of pregnancy but the former is less expensive.Trial registrationTrial registered on http://www.clinicaltrials.gov (NCT01389713)

Outcome of cycles of oocyte in vitro maturation requiring testicular sperm extraction for nonobstructive azoospermia

OBJECTIVE To assess the applicability of oocyte IVM in case of nonobstructive azoospermia (NOA). DESIGN Case series. SETTING Private IVF unit. PATIENT(S) All male partners were affected by NOA. Twenty-one women (20 normo-ovulatory and 1 with polycystic ovary) underwent 27 IVM treatments. The outcome of controlled ovarian stimulation (COS) cycles in normo-ovulatory women whose partners were affected by NOA was assessed for comparison. INTERVENTION(S) Spermatozoa from testicular sperm extraction (TESE) samples were retrieved and cryopreserved before treatment. MAIN OUTCOME MEASURE(S) Rates of fertilization, pregnancy per cycle and transfer, and implantation. RESULT(S) Twenty-seven IVM-TESE cycles were carried out in 21 women. Fertilization rate was 64.4%. In compliance with national legislation, no more than three oocytes were inseminated in each cycle and all developing embryos were transferred. Six single clinical pregnancies were ascertained (22.2% and 27.3% pregnancy rates per cycle and per transfer, respectively). One pregnancy ended in abortion. The others developed to term and gave rise to five healthy babies. Sixty couples underwent standard COS-TESE treatment. Rates of fertilization, clinical pregnancy per cycle, implantation, and abortion were 64.6%, 20.0%, 11.3%, and 16.7%, respectively. Eleven babies were born from ten pregnancies. CONCLUSION(S) Oocyte IVM may represent an option in NOA cases.