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Objective To determine whether the freeze-all policy ensures a higher efficacy in terms of cumulative live birth rate (CLBR) in comparison with a conventional fresh/frozen embryo transfer (ET) approach in patients with normal ovarian response. Design Retrospective, matched, multicenter cohort study. Setting Private IVF centers. Patient(s) This study analyzed 564 completed IVF cycles in which an average of 12–18 oocytes were retrieved. In 435 cycles the conventional strategy was applied, with initial ET followed by frozen embryo replacements, whereas in 129 cycles the freeze-all policy was performed, with elective cryopreservation and deferred use of all viable embryos. Intervention(s) None. Main Outcome Measure(s) The primary endpoint was CLBR. The secondary endpoint was cumulative clinical pregnancy rate. Result(s) Overall, statistically comparable CLBRs were achieved in the fresh/frozen and freeze-all groups (45.5% vs. 53.5%). Stratification of data for age and number of retrieved oocytes confirmed the absence of differences between the two groups. In a subanalysis in which the day of ET and cryopreservation were taken into account, a similar outcome was achieved in cleavage-stage groups (45.6% vs. 46.4%), whereas when ET was performed at the blastocyst stage the CLBR was significantly higher in the freeze-all group (45.3% vs. 66.7%). Conclusion(s) Our CLBR analysis indicates that clinical performance of the freeze-all policy is equivalent to that of the conventional strategy when ET is carried out at the cleavage stage. However, it seems to be superior if associated with cryopreservation and transfer at the blastocyst stage.

Objective To characterize from a vibrational point of view the alterations caused by aging on human oocytes. Design Reproductive biology. Setting Private assisted reproductive technology clinic, synchrotron beam line, and university infrared laboratory. Patient(s) Twenty women of different ages (30 ± 2 and 39 ± 2 years) selected on the basis of detailed inclusion criteria and submitted to controlled ovarian stimulation according to a specific protocol. Intervention(s) Collection of 68 supernumerary oocytes that were not used during the IVF cycle from the above cited consenting patients. Main Outcome Measure(s) Focal Plane Array Fourier transform infrared (FTIR) analysis of human oocytes. Result(s) Specific spectral differences were highlighted in the two experimental groups of oocytes. In particular, in oocytes of 39-year-old women, the occurrence of peroxidative processes and a decrease in the amount of carbohydrates were observed, together with alterations in the phospholipid membrane, proteic pattern, and nucleic acids content. Conclusion(s) For the first time, FTIR spectroscopy was applied to human oocytes, leading to strong evidence of damage from aging in the gametes of mature women, which could be related to a decline in reproductive function. All the information obtained may be considered useful to improve the scientific knowledge on human reproduction and to exploit new strategies for detecting oocyte aging.

Objective To set up a novel protocol of sperm head in vitro decondensation that obviates the problematic effect of the variable degree of sperm chromatin packaging on DNA staining needed for flow cytometric analysis. Design Development of a new cytofluorimetric assay. Setting University laboratory. Patient(s) Semen specimens were obtained from normospermic healthy volunteers at the Department of Life and Environmental Sciences, Universita Politecnica delle Marche. Intervention(s) Setup of the novel in vitro sperm head decondensation protocol; sperm were then stained and analyzed by flow cytometry to measure DNA content. Main Outcome Measure(s) Mean fluorescent channel, DNA content, percentage diploid sperm. Result(s) Native nondecondensed fluorochrome-labeled sperm show significant under-staining, resulting in an underestimated C-value (approximately 1.4 pg). This protocol ensures stoichiometric staining of sperm DNA, which becomes fully reachable by fluorescent probes and makes the diploid (7.12 pg) over haploid (3.56 pg) sperm frequency quantification easier. Conclusion(s) This study establishes a simple method for in vitro sperm head decondensation, which allows accurate detection of the real sperm DNA content.

Objective To update results on outcomes with frozen/thawed oocytes using a differential sucrose concentration during dehydration (0.2 M) and rehydration (0.3 M), combined with a one-step propanediol exposure. Design Retrospective cohort study. Setting Private IVF centers. Patient(s) Infertile couples undergoing IVF treatment. Intervention(s) Oocyte thawing cycles between May 2004 and December 2010. Main Outcome Measure(s) Survival, fertilization, and cleavage rates were reported to evaluate biological outcomes. Clinical pregnancy and implantation rates were analyzed as markers of efficiency. Result(s) Three hundred forty-two patients and 443 cycles were monitored; the survival was 71.8%, fertilization 77.9%, and of the embryos obtained 83.8% were classified as grade 1 and 2. Three hundred ninety-four transfers were performed, resulting in 90 pregnancies. The pregnancy rate per transfer was 22.8% and per patient was 26.3%, with 122 gestational sacs. The implantation rate per embryo was 13.5%. Patients were divided into three groups according to their age: ≤34 years (group A), 35–38 years (group B), and ≥39 years (group C). Biological outcomes were comparable in all three groups, whereas the pregnancy rate per transfer was higher in the first group (27.7% vs. 21.4% and 17.6%). The implantation rates per injected egg were 11.8%, 8.0%, and 7.5% for the three groups, respectively. Conclusion(s) The biological and clinical data obtained on 443 cycles are consistent with our previous results showing that slow freezing of oocytes can be a valid tool in IVF practice when performed with a suitable protocol.

Objective To present a case where recombinant human FSH (r-hFSH) was accidentally used instead of hCG and resulted in oocyte maturation. Design Case report. Setting A tertiary infertility center. Patient(s) A 36-year-old woman underwent IVF for infertility problems. The couple was unable to obtain a pregnancy during the previous 2 years. Intervention(s) Oocyte retrieval after a r-hFSH bolus (two vials of Gonal-F 1050 IU) instead of the routinely used hCG. Main Outcome Measure(s) Oocyte maturity evaluation, fertilization after intracytoplasmic sperm injection, cleavage stage, and embryo quality. Result(s) A total of nine oocytes were retrieved; eight were metaphase II. Three eggs were fertilized by intracytoplasmic sperm injection, and four of the remaining MII oocytes were cryopreserved. All three oocytes fertilized and developed to three good-quality embryos that were transferred on day 2. No pregnancy was obtained from this fresh transfer cycle or from transferred embryos resulting from the thawing cycle. Conclusion(s) A massive r-hFSH administration can lead to the retrieval of mature MII oocytes that are able to fertilize and give good-quality embryos to transfer.

Objective To determine whether Polscope analysis can predict different spindle and chromosome configurations of the oocyte metaphase II (MII) spindle. Design Comparison of Polscope and confocal microscopy analysis of the MII spindle. Setting Private IVF unit. Patient(s) Women undergoing IVF treatment for male or unexplained infertility. Intervention(s) Fresh and frozen-thawed mature oocytes were analyzed through the Polscope and, immediately afterward, fixed for confocal microscopy assessment. Main Outcome Measure(s) Comparison of retardance values, derived from Polscope analysis, between spindles with different microtubule and chromosome configurations, defined by confocal microscopy evaluation. Measurements of spindle longitudinal axis through the Polscope and confocal microscopy. Result(s) The mean retardance values of different categories of spindle configuration were not statistically significant in almost all cases, allowing only the identification of spindles with highly disorganized microtubules and chromosomes in frozen-thawed oocytes. In spindles with bipolar organization, the Polscope produced measurements of the spindle main axis which were in all cases statistically smaller compared with confocal microscopy evaluation. Conclusion(s) Retardance measurements have limited predictive value of the degree of spindle fiber order and chromosome position in routine clinical settings. Also, under the conditions tested, morphometric evaluation of the spindle through the Polscope is not consistent with confocal analysis. This suggests that the Polscope may still be a rather inefficient method for assessing the metaphase II spindle and, as a result, for noninvasive oocyte selection.

Objective To evaluate the role of 3,3′,5-triiodothyronine (T 3 ) on ovarian follicle development in vitro. Design Experimental study. Setting University reproductive biology unit. Animal(s) CD1 mice (female). Intervention(s) In vitro culture. Main outcome measure(s) Assessment of follicle and oocyte development following a 6-day culture with FSH (100 mU/mL), (Bu) 2 cAMP (0.5 mM), and T 3 (1–100 nM), alone or in combination. Result(s) A high percentage of in vitro grown (IVG) FSH-stimulated follicles formed an antral cavity (AC), while the addition of T 3 (100 nM) significantly reduced this response in a statistically significant manner. A statistically significant recovery of AC formation was obtained by limiting exposure to 4 days of culture. Formation of AC was induced by (Bu) 2 cAMP, which prevented T 3 -mediated suppression of AC formation. Under different conditions, high proportions of germinal vesicle-arrested IVG oocytes displayed condensed chromatin configuration. The capacity to undergo germinal vesicle breakdown (GVBD) was similar in all groups, and a statistically significant reduction in the percentage of oocytes with PB1 was recorded when T 3 was added to FSH or (Bu) 2 cAMP. This ability was partially recovered by removing T 3 on day 4 of culture. Conclusion(s) Exposure to high T 3 concentrations can impair preantral follicle development, but this effect can be partly reverted by restoring a physiological hormonal milieu before follicle commitment to antral development.

Objective: To test the efficacy of endometrial preparation with exogenous steroids, without pretreatment with gonadotropin-releasing hormone (GnRH) agonist, in women with normal ovarian function. Design: Prospective randomized study. Setting: Private outpatient infertility clinic. Patient(s): Two hundred ninety-six women undergoing frozen-thawed embryo transfer. Intervention(s): In group 1 (146 patients), depot GnRH agonist was administered in the luteal phase; treatment with 17β-estradiol transdermal patches at steadily increasing dosage from 100 to 300 μg was then given for at least 12 days. In group 2 (150 patients), endometrial preparation began on day 1 of menstrual cycle. The starting dose was 200 μg; this was increased to 300 μg after 7 days. Main Outcome Measure(s): Pregnancy, abortion, implantation and cancellation rates. Result(s): In group 2, six cycles (4%) were cancelled due to evidence of ovulation. Groups were similar in the percentage of embryos that survived freezing-thawing (77.1% in group 1 and 76.6% in group 2) and in the number of embryos transferred per patient (2.1 ± 0.6 and 2.1 ± 0.7, respectively). Groups 1 and 2 did not differ significantly in rates of pregnancy (19.7% and 24.1%), abortion (17.8% and 11.7%), and implantation (10.4% and 11.9%). Conclusion(s): Endometrial preparation for frozen-thawed embryo transfer based exclusively on steroid administration appears to be as effective as the more conventional protocol involving preliminary desensitization with a GnRH agonist. This simplified protocol reduces costs, minimizes pharmacologic treatment, and increases patient compliance.

Objective To investigate if the presence of an abnormally large follicle during controlled ovarian hyperstimulation (COH) under pituitary suppression has any effect on the outcome of the cycle. Design Prospective, observational. Setting The reproductive endocrinology unit of a university hospital. Patients One hundred fifty patients undergoing COH for assisted reproductive techniques during a 6-month period (August 1990 to January 1991). Interventions Transvaginal follicular aspiration and IVF-uterine ET. Results A total of 19cycles had abnormally large follicles identified on day 8 of the stimulation cycle after normal baseline ultrasound. Gonadotropins were continued and hCG injection was indicated when two or more follicles of the main cohort achieved a diameter of 20 to 22mm. Twenty preovulatory oocytes were retrieved from 22 large follicles. Two were transferred for GIFT and 18 were inseminated in vitro, resulting in a 72.0% fertilization rate. The mean number of oocytes retrieved per patient was 10.9, 71.4% of which were mature with a fertilization rate of 67.7%. All these figures were comparable with the results obtained in the 131 patients of the control group undergoing IVF. No evidence of premature luteinization was observed in the study group, based on plasma P levels (×0.83ng/mL [conversion factor to SI unit, 3.180], range 0.31 to 1.40ng/mL). The clinical pregnancy rate for the group with abnormally large follicles did not differ from the control group (27.8% versus 28.2%, respectively). Conclusions The presence of an abnormally large follicle during COH under pituitary suppression does not affect the outcome of the cycle. Moreover, under these conditions, continuous gonadotropin stimulation of a follicle to diameters considerably larger than the standard ones does not have a detrimental effect on the oocyte contained in it, suggesting that oocyte aging is an independent process from follicular growth once LH surge is prevented.

Objective To determine the potential of aging uterus in terms of pregnancy, implantation and abortion rates, and obsteric complications in postmenopausal women age 50 and over, receiving oocyte donation. Design Retrospective data analysis. Setting A tertiary infertility center. Patients Thirty-four postmenopausal women (50 to 62 years of age) undergoing a total of 61 cycles of oocyte donation. Interventions Hormone replacement therpy(HRT) was given using increasing doses of 17 β -E 2 (2,4, and 6 mg) and 100 mg of P in oil. Main Outcome Measure Pregnancy, abortion, and implantation rates, and pregnancy outcome. Results There were 34 patients treated through 61 cycles. Fifty-five transfer cycles were performed. A totl of 18 clinical pregnancies were achieved with a pregnancy rate per tranfer of 32.7% and per patient of 52.9%. There was one abortion, resulting in an abortion rate of 5.5%. Twenty-one of the 116 transferred embroys implanted, resulting in an implantation rate of 18%. No statistically different rates were found in oocyte donors. Conclusion Women over the age of 50 and up to 62 can become pregnant using donated oocytes. The aging uterus after HRT allows implantation as well as in young women, and it is able to carry pregnancy to term apparently without any problem.