Lucia De Santis

Polar body morphology and spindle imaging as predictors of oocyte quality

It has been suggested that first polar body (PBI) morphology reflects oocyte competence. Oocytes with an intact normal-sized PBI have been described as generating better day 2 embryos, higher blastocyst yield, and increased pregnancy and implantation rates. In other studies, PBI morphology was found to be unrelated to fertilization rate, embryo quality, and blastocyst formation. In a prospective analysis, the predictive value of the PBI was investigated by comparing the development of oocytes retrieved from intracytoplasmic sperm injection patients and displaying different PBI morphology, classified according to the following characteristics: normal size and smooth surface (I), fragmented (II), rough surface (III), or large size (IV). Fertilization rates were 59, 57, 64 and 60% respectively. No significant differences were found between the various groups. The proportions of high quality (grade A) day 2 embryos were also comparable among groups I-III (14, 12 and 17% respectively), while the low number of grade A embryos in group IV (two embryos) did not allow comparison with the other classes. These data do not suggest that PBI selection can contribute to identification of embryos with high developmental ability. In order to establish alternative criteria for oocyte selection, a metaphase II (MII) spindle analysis was also conducted via Polscope. In oocytes of patients of different age, spindle retardance (which reflects the high order and density of microtubules) was compared with parameters of embryo development. In aged patients, a trend was observed between low retardance and poor embryo quality, although in general the association between retardance and oocyte developmental performance did not reach statistical significance.

Myo-inositol in patients with polycystic ovary syndrome: A novel method for ovulation induction

Background. Polycystic ovary syndrome (PCOS) is often characterized by chronic oligo- or anovulation (usually manifested as oligo- or amenorrhea), and hyperandrogenism. In addition, 30–40% of PCOS women have impaired glucose tolerance, and a defect in the insulin signaling pathway (inositol-containing phosphoglycan mediators) seems to be implicated in the pathogenesis of insulin resistance. PCOS patients are subfertile as a consequence of such ovulatory disorders and often need drugs, such as clomiphene citrate or follicle-stimulating hormone, for ovulation induction, which increases the risk of multiple pregnancy and ovarian hyperstimulation syndrome. We hypothesized that the administration of an isoform of inositol (myo-inositol), belonging to the vitamin B complex, would improve the insulin-receptor activity, restoring normal ovulatory function. Materials and methods. Twenty-five PCOS women of childbearing age with oligo- or amenorrhea were enrolled in the study. Ovulatory disorder due to PCOS was apparently the only cause of infertility; no tubal defect or deficiency of male semen parameters was found. Myo-inositol combined with folic acid (Inofolic®) 2 g twice a day was administered continuously. During an observation period of 6 months, ovulatory activity was monitored with ultrasound scan and hormonal profile, and the numbers of spontaneous menstrual cycles and eventually pregnancies were assessed. Results. Twenty-two out of the 25 (88%) patients restored at least one spontaneous menstrual cycle during treatment, of whom 18 (72%) maintained normal ovulatory activity during the follow-up period. A total of 10 singleton pregnancies (40% of patients) were obtained. Nine clinical pregnancies were assessed with fetal heart beat at ultrasound scan. Two pregnancies evolved in spontaneous abortion. Conclusion. Myo-inositol is a simple and safe treatment that is capable of restoring spontaneous ovarian activity and consequently fertility in most patients with PCOS. This therapy did not cause multiple pregnancy.

Myo-inositol may improve oocyte quality in intracytoplasmic sperm injection cycles. A prospective, controlled, randomized trial.

OBJECTIVE To determine the effects of myo-inositol on oocyte quality in polycystic ovary syndrome (PCOS) patients undergoing intracytoplasmic sperm injection (ICSI) cycles. DESIGN A prospective, controlled, randomized trial. SETTING Assisted reproduction centers. PATIENT(S) Sixty infertile PCO patients undergoing ovulation induction for ICSI. INTERVENTION(S) All participants underwent standard long protocol. Starting on the day of GnRH administration, 30 participants received myo-inositol combined with folic acid (Inofolic) 2 g twice a day and 30 control women received folic acid alone, administrated continuously. MAIN OUTCOME MEASURE(S) Primary end points were number of morphologically mature oocytes retrieved, embryo quality, and pregnancy and implantation rates. Secondary end points were total number of days of FSH stimulation, total dose of gonadotropin administered, E(2) level on the day of hCG administration, fertilization rate per number of retrieved oocytes, embryo cleavage rate, live birth and miscarriage rates, cancellation rate, and incidence of moderate or severe ovarian hyperstimulation syndrome. RESULT(S) Total r-FSH units (1,958 +/- 695 vs. 2,383 +/- 578) and number of days of stimulation (11.4 +/- 0.9 vs. 12.4 +/- 1.4) were significantly reduced in the myo-inositol group. Furthermore, peak E(2) levels (2,232 +/- 510 vs. 2,713 +/- 595 pg/mL) at hCG administration were significantly lower in patients receiving myo-inositol. The mean number of oocytes retrieved did not differ in the two groups, whereas in the group cotreated with myo-inositol the mean number of germinal vesicles and degenerated oocytes was significantly reduced (1.0 +/- 0.9 vs. 1.6 +/- 1.0), with a trend for increased percentage of oocytes in metaphase II (0.82 +/- 0.11% vs. 0.75 +/- 0.15%). CONCLUSION(S) These data show that in patients with PCOS, treatment with myo-inositol and folic acid, but not folic acid alone, reduces germinal vesicles and degenerated oocytes at ovum pick-up without compromising total number of retrieved oocytes. This approach, reducing E(2) levels at hGC administration, could be adopted to decrease the risk of hyperstimulation in such patients.

Multicenter observational study on slow-cooling oocyte cryopreservation: clinical outcome

OBJECTIVE To evaluate the efficacy of oocyte cryopreservation by a single slow-cooling protocol involving sucrose (0.2 mol/L) in the freezing solution. DESIGN Observational comparison of the clinical outcome in fresh and frozen thawed cycles. SETTING Public and private IVF centers. PATIENT(S) Infertile couples undergoing IVF treatment. INTERVENTION(S) Use of a maximum three oocytes in fresh cycles, as established by local law, and cryopreservation and later use of surplus oocytes. Likewise fresh cycles, maximum three thawed oocytes were used per cycle. All thawed oocytes were microinjected. MAIN OUTCOME MEASURE(S) Embryologic and clinical parameters of fresh and thawed cycles. RESULT(S) Two thousand forty-six patients underwent 2,209 oocyte retrievals involving oocyte cryopreservation. Overall, the survival rate of thawed oocytes was 55.8%. In 940 thaw cycles, the mean numbers of inseminated oocytes and fertilization rates were significantly decreased vs. fresh cycles outcomes (2.6 ± 0.7 vs. 2.9 ± 0.2 and 72.5% vs. 78.3%, respectively), as were the rates of implantation (10.1% vs. 15.4%), pregnancy rates per transfer (17.0% vs. 27.9%), and pregnancy rates per cycle (13.7% vs. 26.2%). Differences in clinical outcome were found among centers. A pregnancy rate per thawing cycle above 14% was achieved by most clinics. Fifty-seven retrievals involving oocyte cryopreservation achieved a pregnancy after fresh embryo replacement. Implantation and pregnancy rates per embryo transfer and per thawing cycles were 17.5%, 28.6%, and 24.6%, respectively. CONCLUSION(S) Under the conditions tested, the clinical outcome of oocyte slow-cooling cryopreservation is reduced compared with fresh cycles. Nevertheless, in cases of inapplicability of embryo cryopreservation, oocyte cryopreservation should be offered to patients with surplus oocytes.

Pathologic findings in hysteroscopy before in vitro fertilization-embryo transfer (IVF-ET)

Background. The aim of this study was to evaluate hysteroscopy routinely performed prior to in vitro fertilization-embryo transfer (IVF-ET). Methods. We analyzed in a prospective study 300 patients who underwent hysteroscopy before the first IVF-ET cycle. We analyzed then in a retrospective manner 300 patients who did not perform hysteroscopy. Results. One-hundred-and-eighty (60%) hysteroscopies were normal but 120 (40%) revealed an unsuspected intrauterine abnormality. We did not find statistically significant differences between patients with normal or abnormal hysteroscopy in any characteristic. We found a statistically significant difference in pregnancy rate between women who performed hysteroscopy before IVF-ET cycle and in women who did not perform it. Conclusions. Hysteroscopy, as a routine examination, should be performed before the first IVF-ET cycle in all patients.

Natural cycle as first approach in aged patients with elevated follicle-stimulating hormone undergoing intracytoplasmic sperm injection: a pilot study.

Background. Poor ovarian response to standard in vitro fertilization–embryo transfer (IVF-ET) protocols or different regimens of treatment, as consequence of a diminished ovarian reserve, correlates strictly with patient age, elevated follicle-stimulating hormone (FSH) and reduced antral follicle count. The aim of the present pilot study was to evaluate the outcome of patients with poor prognostic features undergoing IVF-ET with natural cycles as a first approach and not as a consequence of a previous failure treatment. Materials and methods. Eighteen aged patients (mean ± standard deviation 40.2 ± 0.7 years, range 37–43 years) with elevated serum FSH and reduced antral follicle count underwent intracytoplasmic sperm injection (ICSI) after spontaneous ovulation. Results. A total of 26 natural cycles with ICSI were analyzed. Pregnancy was observed in three patients, of which two were ongoing as assessed by fetal heart beat at ultrasound scan performed 4–5 weeks after ET. Conclusion. The overall pregnancy rates achieved (11.5% per cycle, 20.0% per ET) are comparable with those of conventional IVF-ET in aged patients, and not impaired by a single embryo transferred. Better embryo quality, as a consequence of natural selection of oocytes, better endometrium receptivity and monthly repeatability of the procedure, can balance the relatively low chance to perform ET.

Consecutive cycles in in vitro fertilization-embryo transfer

Background. The decline of female fertility with advancing age is well documented. The aim of this study was to compare the ovarian performance after repeated ovarian stimulation cycles in women of different ages. Methods. Four hundred patients who started at least three in vitro fertilization (IVF) cycles during the 5-year period between 1998 and 2002 were identified. The patients were divided into four groups: the 25–30 age group (n = 90), the 31–35 age group (n = 150), the 36–40 age group (n = 110) and the 41–45 age group (n = 50). Results. Comparing subsequent cycles versus the first treatment cycle we found a statistically significantly increased number of ampules of recombinant follicle stimulating hormone (rFSH) needed to reach follicles maturation (p < 0.001). The number of ampules of gonadotropin required was significantly higher (p < 0.001) in the groups of advanced age compared with the groups of young women. For women in the 36–40 group and in the 41–45 group we found the number of follicles, the number of oocytes and the proportion of grade A embryos, in every cycle, were significantly lower than in the groups of young women. We compared the characteristics of ovarian stimulation and response of a single age group in different consecutive cycles. We found significant differences (p < 0.05) only in the number of ampules required. Conclusions. Maternal age adversely affected ovarian performance. During repeated IVF cycles we also noted an age-independent decline of ovarian response.

Theoretical and experimental basis of slow freezing

In human IVF, cryopreservation of oocytes has become an alternative to embryo storage. It has also shown enormous potential for oocyte donation, fertility preservation and animal biotechnology. Mouse oocytes have represented the elective model to develop oocyte cryopreservation in the human and over several decades their use has made possible the development of theoretical and empirical approaches. Progress in vitrification has overshadowed slow freezing to such an extent that it has been suggested that vitrification could soon become the exclusive cryopreservation choice in human IVF. However, recent studies have clearly indicated that human embryo slow freezing, a practice considered well established for decades, can be significantly improved by a simple empirical approach. Alternatively, recent and more advanced theoretical models can predict oocyte responses to the diverse factors characterizing an entire slow-freezing procedure, offering a global method for the improvement of current protocols. This gives credit to the notion that oocyte slow freezing still has considerable margins for improvement.

Hypogonadotropic hypogonadism in a trisomy X carrier: phenotype description and genotype correlation

Abstract We report on a 31-year old female who presented at genetic counseling for a small uterus, secondary amenorrhea and sterility. Gonadotropic hormone levels were low, suggesting a Hypogonadotropic Hypogonadism (HH) condition. Cytogenetic analysis demonstrated the presence of Trisomy X associated to an interstitial deletion of chromosome 4q13.2, resulting in the complete loss of a copy of the GNRHR gene. As GNRHR is known to be responsible for an autosomal recessive form of HH, we checked the status of the undeleted allele and we found the Q106R substitution. In conclusion, the results of our cytogenetic and molecular analyses have allowed us to clarify the etiology of the patients condition.

Slow Freezing of Oocytes

Oocyte cryopreservation has several potential applications in human assisted reproduction technology, such as to maximize cycle cumulative outcome, limit the number of embryos generated for fresh embryo transfer, and support programs of fertility preservation and oocyte donation. Mature oocytes are intrinsically more susceptible to cryodamage compared with embryos. Fully grown immature oocytes are even less amenable to cryopreservation, due to the necessity to preserve morpho-functional relationship with companion cumulus cells. Consequently, not surprisingly, initial attempts aimed at cryostoring oocytes with conventional controlled rate slow-cooling (CRSC) protocols were frustrated by low survival rates and poor clinical outcomes. Several studies have confirmed that cryopreservation, if performed with suboptimal protocols, generates diverse types of cell damage, such as zona pellucida rupture, release of cortical granules, ultrastructural damage, and alterations in cell cycle regulation. Notwithstanding, perseverant and systematic research efforts have led to improved slow-cooling protocols that, although unable to increase survival rates above 75–80%, can achieve clinical outcomes that, if assessed in terms of number of implantations per number of thawed oocytes, are comparable with those obtained by vitrification. Younger patients seem to benefit particularly from oocyte CRSC, while results from older women are rather disappointing even beyond the expected effect of female age. The health of babies derived from oocytes stored by CRSC has been investigated. Relevant studies are few and numerically limited but do not suggest a health impact of cryopreservation. Overall, over the last decade, oocyte cryopreservation by CRSC has been replaced by vitirification. However, some intrinsic advantages of CRSC should not be overlooked, such as a higher reproducibility of protocols and automated monitoring of cooling phases.