Embryos with morphokinetic abnormalities may develop into euploid blastocysts.

Irregular cleavage divisions are expected to produce chromosomally deviant embryos. We investigated whether embryos from irregular cleavages could develop into euploid blastocysts, and, if so, whether any evidence existed of a self-correction mechanism of the embryo. We also investigated the role of different dynamic aspects of morula compaction in this process. A total of 791 embryos from 141 patients undergoing pre-implantation genetic screening were retrospectively analysed using a time-lapse imaging system, and multiple cell divisions were evaluated. A total of 276 embryos developed into blastocysts suitable for biopsy and chromosome screening through array-comparative genomic hybridization. As well as testing trophectoderm biopsy specimens for aneuploidy, excluded cells of 18 blastocysts, which developed from partially compacted morulas, were also analysed. Unique data on the developmental fate of embryos with cleavage abnormalities are presented, and a potential mechanism of 'aneuploidy rescue' is postulated through which mosaic embryos may form partially compacted morulas to exclude aneuploid cells. In addition, this process seems to be less efficient in older women. The data obtained also provide further evidence that excluded cells should not be used to infer the cytogenetic status of the embryo.

Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop.

This study describes and compares the possible effects of vitrification on the ultrastructural morphology of 20 human mature oocytes vitrified using two different supports, cryoleaf (n = 10) and cryoloop (n = 10). Fresh human mature oocytes (n = 15) were used as controls. Fresh and vitrified-warmed oocytes appeared rounded, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Sparse microvacuolization was only occasionally detected in fresh and vitrified-warmed oocytes, to the same extent. About 50% of the vitrified oocytes contained atypical, small and slender mitochondria-smooth endoplasmic reticulum aggregates, whereas a non-homogeneous microvillar pattern was observable in only 30% of the oocytes subjected to vitrification, regardless of the support utilized. Cortical granule content appeared generally reduced after vitrification, but cryoleaf-supported oocytes contained more cortical granules than cryoloop-supported oocytes (P < 0.05). Thus good overall preservation and virtual absence of cytoplasmic vacuolization seem to be the most relevant markers of quality in vitrified-warmed oocytes, using either support. In addition, cryoleaf-supported oocytes retained a higher number of cortical granules than cryoloop-supported oocytes. The variety of ultrastructural alterations recorded emphasizes the need for further studies aimed at assessing the actual tolerance of human oocytes to vitrification.

Vitrification may increase the rate of chromosome misalignment in the metaphase II spindle of human mature oocytes.

The metaphase II (MII) spindle of the human oocyte may be damaged by cryopreservation. High performance confocal microscopy was used to assess meiotic spindle and chromosome organization in oocytes after vitrification by the cryoleaf system. Three hours after retrieval, donor mature oocytes were fixed or vitrified. Vitrification was performed by equilibration in 7.5% ethylene glycol (EG) and 7.5% dimethylsulphoxide (DMSO), transfer to 15% EG, 15% DMSO and 0.5 mol/l sucrose, and loading onto cryoleaf strips. Tubulin staining was found in all survived vitrified-warmed oocytes, the majority (62.8%) of which displayed a bipolar spindle. A normal bipolar spindle configuration and equatorial chromosome alignment was observed only in a part of vitrified-warmed oocytes (32.6%). This frequency was significantly lower in comparison to fresh oocytes (59.1%). In another fraction of vitrified-warmed oocytes (30.2%), spindle bipolarity was associated to one or more non-aligned scattered chromosomes that often appeared tenuously associated with the lateral microtubules of the spindle. Furthermore, in cryopreserved oocytes with a bipolar spindle, a significantly increased pole-to-pole distance (14.9 +/- 2.3 microm) was found in comparison to the fresh control (12.4 +/- 2.6 microm) (P = 0.001). Therefore, under the conditions tested, vitrified-warmed oocytes maintain a MII spindle with a bipolar organization. However, chromosome alignment appears to be partly compromised.

A PolScope evaluation of meiotic spindle dynamics in frozen-thawed oocytes.

In mature human oocytes, the metaphase II (MII) spindle presence and birefringence signal detected through the PolScope may vary before and after freezing. In particular, spindle dynamics during the first few hours after thawing is still under study. In this study, oocytes from stimulated ovaries were cryopreserved in 1.5 mol/l 1,2-propanediol with 0.3 mol/l sucrose using a slow freezing-rapid thawing method. Oocytes were examined with the PolScope for the presence, intensity of signal birefringence and size of the meiotic spindle before freezing and at 0, 1 and 2 h post-thaw (where 0 h = the time of the end of the thawing procedure). Of the 173 surviving oocytes exhibiting a spindle before freezing, 82.7% (143/173) showed spindle birefringence within 1 h of thawing. However, at the end of the thawing procedure the intensity of spindle birefringence (retardance) and the spindle length were smaller in comparison to the pre-freezing condition. These parameters increased after 1 h, although were not restored to the value observed before freezing. No significant changes were observed by extending the culture to 2 h.

Meiotic spindle dynamics in human oocytes following slow-cooling cryopreservation.

BACKGROUND: The demand for cryopreservation of human oocytes is increasing in assisted reproduction clinics and yet remains an experimental procedure. Surprisingly, little is known about the effects of cryopreservation on spindle-chromosome interactions and the recovery of meiotic spindle functionality. The goal of these studies was to evaluate the process of meiotic spindle reassembly and chromosome alignment in cryopreserved human metaphase II oocytes. METHODS: Unfrozen control oocytes were compared with frozen oocytes fixed at 0, 1, 2 and 3 h after thawing. Oocytes were analysed by confocal microscopy and subjected to 3-dimensional image analysis to evaluate spindle integrity. RESULTS: Freezing resulted in a loss of spindle bipolarity and chromosome alignment. One hour following thawing, most oocytes recovered spindle bipolarity and equatorial chromosomal alignment. However, between 2 and 3 h, a progressive loss of chromosome alignment was observed. Further analysis revealed a positive correlation between spindle length and number of displaced chromosomes following freezing. This time-dependent redistribution of chromosomes involved outward displacement from the equatorial plate and retention at the surface of the meiotic spindle. CONCLUSIONS: Spindle disassembly incurred by cryopreservation is rapidly reversed and is coordinated with chromosome alignment within 1 h but is not sustained at later times.

Freezing spermatozoa obtained by testicular fine needle aspiration: a new technique.

Testicular fine needle aspiration (TEFNA) of spermatozoa in azoospermic patients in advance of intracytoplasmic sperm injection could be useful to avoid the possibility of no recovery of spermatozoa on the day of oocyte retrieval. The conventional freezing procedure for these spermatozoa is not appropriate because of their very low number and poor in-situ motility. This article presents a new procedure for the freezing of TEFNA-recovered spermatozoa. A total of 1063 spermatozoa (10-340 cells/sample) were frozen by this method for research purposes. Before freezing, 13.7% were motile. The recovery rate after thawing was 100%. After thawing, 3.6% motility was observed. In a separate study group, the total number of frozen spermatozoa was 431 (2-300 cells/sample). Before freezing, the sperm motility rate was 3.5%. After thawing, 100% of the spermatozoa were retrieved with a motility rate of 2.3%. One biochemical pregnancy was obtained. The procedure yielded excellent recovery and good motility rates after thawing. However, because of the low number of cases, any conclusion about the efficiency of the technique is premature.

Evidence-based clinical outcome of oocyte slow cooling.

In the last few years, there has been a significant improvement in oocyte cryopreservation techniques. To investigate the clinical significance of oocyte freezing, an assessment of the cumulative pregnancy rate per started cycle derived from the use of fresh and frozen-thawed oocytes was performed. Between 2004 and 2006, 749 cycles were carried out, in which no more than three fresh oocytes were inseminated either by standard IVF or microinjection. Supernumerary mature oocytes were cryopreserved by slow cooling. Cryopreservation of fresh embryos was performed in rare cases to prevent the risk of ovarian hyperstimulation syndrome using a standard embryo freezing protocol. Fresh embryo transfer cycles totalled 680, 257 of which resulted in pregnancy. The pregnancy rates per patient and per transfer were 34.3% and 37.8% respectively. When frozen-thawed oocytes were used, following 660 thawing cycles, 590 embryo transfers were performed in 510 patients. Eighty-eight pregnancies were achieved with embryos from frozen oocytes, with a success rate of 17.2% per cycle. When fresh and frozen-thawed cycles were combined, the number of pregnancies was 355, giving a cumulative pregnancy rate of 47.4%. Oocyte cryopreservation can contribute considerably to the overall clinical success, ensuring a cumulative rate approaching that achievable with embryo storage.

Truths and myths of oocyte sensitivity to controlled rate freezing.

The mammalian oocyte is especially sensitive to cryopreservation. Because of its size and physiology, it can easily undergo cell death or sub-lethal damage as a consequence of intracellular ice formation, increase in the concentration of solutes and other undesired effects during the conversion of extracellular water into ice. This has generated the belief that oocyte storage cannot be achieved with the necessary efficiency and safety. However, many concerns raised by oocyte freezing are the result of unproven hypotheses or observations conducted under sometimes inappropriate conditions. For instance, spindle organization can undergo damage under certain freezing conditions but not with other protocols. The controversial suggestion that cryopreservation induces cortical granule discharge and zona pellucida hardening somehow questions the routine use of sperm microinjection. Damage to mouse oocytes caused by solute concentration is well documented but, in the human, there is no solid evidence that modifications of freezing mixtures, to prevent this problem, provide an actual advantage. The hope of developing oocyte cryopreservation as a major IVF option is becoming increasingly realistic, but major efforts are still required to clarify the authentic implications of oocyte cryopreservation at the cellular level and identify freezing conditions compatible with the preservation of viability and developmental ability.

Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration.

BACKGROUND: Recently, interest in oocyte cryopreservation has steadily increased. Newly developed protocols have dramatically improved survival rates, removing perhaps the major hurdle that has prevented this approach from becoming a fully established form of treatment. However, the clinical efficiency of these protocols has not been exhaustively explored and therefore remains controversial. METHODS: Morphologically normal oocytes displaying the first polar body were frozen-thawed with a slow cooling protocol that utilized 1.5 mol/l propane-1,2-diol (PrOH) and 0.3 mol/l sucrose. RESULTS: A total of 927 oocytes from 146 patients were frozen-thawed, achieving a 74.1% survival rate. Over 76% of microinjected oocytes displayed two pronuclei 16 h post-insemination, while the proportion of embryos at 44-46 h post-insemination was 90.2%. At this time point, the majority (68.3%) of embryos were at the two-cell stage, showing in most cases (78.7%) minimal or moderate fragmentation. Eighteen clinical pregnancies, three of which were twin, were observed, giving rise to rates of 12.3 and 9.7%, calculated per patient and per embryo transfer, respectively. The implantation rate was 5.2%. To date, four children have been born and three pregnancies resulted in spontaneous abortions, while the remaining pregnancies are ongoing. CONCLUSIONS: Our data indicate that although the combination of slow cooling and high sucrose concentration ensures high rates of oocyte survival, it is not sufficient to guarantee a high standard of clinical efficiency.

Perifollicular vascularity and its relationship with oocyte maturity and IVF outcome.

New markers of embryo ability to implant are pursued continuously. Understanding whether an oocyte is really "mature," that is, ready to be fertilized, would be of great help in choosing an embryo that will implant. It is usual to pay attention to the phase of meiosis, considering the extrusion of the polar body (metaphase II) to be the only sign of the maturity of the oocytes. Nevertheless, understanding more about how the cytoplasm contributes to an oocyte's competency also shows promise as a method of predicting which embryos will implant. Some studies about perifollicular vascularity have demonstrated that embryos originating from oocytes developed in well-vascularized follicles have a higher implantation rate than those originating from oocytes developed in follicles with poor vascularization. Here, we report our results from a preliminary study in which embryos were transferred according to the degree of vascularization of the follicle. Women who received embryos originating from oocytes developed in well-vascularized follicles had a statistically higher pregnancy rate than women who received embryos deriving from oocytes grown in more poorly vascularized follicles (34% vs. 13.7%).

Alcohol consumption and total estradiol in premenopausal women.

The present paper analyzes the relation between alcohol intake and serum total estradiol in premenopausal women while attempting to control or reduce several sources of variability of serum estradiol. Sixty premenopausal women were recruited, and alcohol intake was estimated by a semiquantitative questionnaire. Interviews, anthropometric measurements, and blood drawings (after overnight fasting) were conducted twice, 1 year apart. Both blood samples were obtained on the same day of the luteal phase of the cycle, in the same month and in the same hour and minute of the day. Samples from the first drawing were stored at -80 degrees C. Serum from both drawings was assayed simultaneously and in blind fashion. A significant association between alcohol intake and estradiol was found when estradiol was averaged across the two visits (Spearman's r = 0.29; P < 0.05). To control for intraindividual variability of estradiol over time, participants were then divided into tertiles of hormone distribution for each of the two sets of measurements and classified based on their consistency in estradiol across the two visits. Women showing consistently high estradiol levels at both visits were characterized by a significantly higher alcohol intake (92.8 g/week) in comparison with those showing consistently low estradiol at both visits (31.6 g/week). Furthermore, the prevalence of drinkers in the group with consistently high estradiol was significantly higher than in the group with consistently low estradiol. The present report indicates that drinkers seem to be characterized by consistently higher estradiol than nondrinkers, and that when the variability of estradiol in premenopause is considered, it is possible to identify a relationship between alcohol intake and estradiol.

Reliability of serum hormones in premenopausal and postmenopausal women over a one-year period.

Serum hormones have been intensively investigated in association with several chronic diseases, but limited information exists on the reliability of a number of hormone determinations. The one-year reproducibility of dehydroepiandrosterone sulfate (DHEAS), total and free testosterone, total estradiol, insulin, C-peptide, and prolactin was studied in 60 premenopausal and 47 postmenopausal women recruited in Varese province, Italy, 1991-1992. The hormonal determinations were made in blood samples collected twice, one year apart, after 12-h fast, in the same month, day, and hour and for premenopausal women on the same day of the luteal phase of the menstrual cycle. Samples from the first drawing were stored at -80 degrees C. Samples from both drawings were assayed simultaneously and in blind fashion. Total estradiol in postmenopause was not evaluated for limitation in the sensitivity of the laboratory method. The intraclass correlation coefficient in premenopausal women was 0.85 for DHEAS, 0.60 for total testosterone, 0.66 for free testosterone, 0.81 for insulin, 0.83 for C-peptide, 0.40 for prolactin, and 0.06 for total estradiol. In postmenopausal women, the coefficient was 0.90 for DHEAS, 0.88 for total testosterone, 0.71 for free testosterone, 0.67 for insulin, 0.73 for C-peptide, and 0.18 for prolactin. These data indicate that total estradiol measured during the luteal phase has a poor intraindividual reproducibility over time, and these findings may have important implications in studies of hormones in the etiology of chronic disease.

Serum sex hormone levels after menopause and subsequent breast cancer.

BACKGROUND: High levels of androgens and estrogens have been reported to be associated with breast cancer. However, the multiplicity of factors that influence hormone levels and methodologic issues complicate the study of the relationship between steroid sex hormones and breast cancer. PURPOSE: Using an improved study design, we assessed prospectively the relationship between the principal steroid sex hormones in serum and the subsequent occurrence of invasive breast cancer in postmenopausal women. METHODS: Four thousand fifty-three healthy postmenopausal women aged 40-69 years, were enrolled from June 1987 through June 1992 in a prospective investigation of hormones and diet in the etiology of breast tumors (ORDET study) as part of a larger volunteer cohort of 10 788 premenopausal and postmenopausal women from Varese Province, northern Italy. At recruitment, blood samples were taken between 8:00 AM and 9:30 AM (after overnight fasting), and sera were preserved in -80 degree Celsius freezers. Women who had received hormone treatment in the 3 months prior to enrollment, who had bilateral ovariectomy, or who had a history of cancer or liver disease were not recruited. Twenty-five women in the final eligible cohort of postmenopausal women developed histologically confirmed, invasive breast cancer during the first 3.5 years of follow-up for the cohort (13 537 women-years). For each case subject, four control subjects were randomly chosen after matching for factors possibly affecting hormone preservation in serum. One case subject and eight control subjects were excluded because premenopausal hormonal patterns were found; thus, after also excluding the four control subjects matched to the ineligible case subject, we included 24 case and 88 control subjects. In the spring of 1994, stored sera of case and control subjects were assayed in a blinded manner for dehydroepiandrosterone sulfate and estradiol (E2) by in-house radioimmunoassay and for total and free testosterone and sex hormone-binding globulin by commercially available nonextraction iodination kits. Mean differences in risk factors were tested by analysis of variance for paired data. Relative risks (RRs) were estimated by conditional logistic regression analysis. All P values resulted from two-sided tests. RESULTS: Age-adjusted mean values of total testosterone, free testosterone, and E2 were significantly higher in case subjects than in control subjects: total testosterone, 0.34 ng/mL versus 0.25 ng/mL (P<.001); free testosterone, 1.07 pg/ml versus 0.77 pg/mL (P= .006); and E2, 25 pg/mL versus 22 pg/mL (P= .027). Age-adjusted RRs for breast cancer in increasing tertiles were as follows: for total testosterone, 1.0, 4.8, and 7.0 (P for trend =.026); for free testosterone, 1.0, 1.8, and 5.7 (P for trend=.005); and for total E2, 1.0, 7.1, and 5.5 (P for trend= .128). CONCLUSIONS AND IMPLICATIONS: This prospective study provides further evidence in support of the already established association between elevated estrogen levels and breast cancer. Even more importantly, it provides new evidence that high serum testosterone levels precede breast cancer occurrence.

Validity for epidemiological studies of long-term cryoconservation of steroid and protein hormones in serum and plasma.

Prospective studies based on the storage of biological samples at low temperature have opened new perspectives in etiological research on cancer. In planning these studies a crucial question is to evaluate whether the long-term preservation of samples is able to affect the categorization of the subjects involved. In the frame of the ORDET project, a prospective study of hormones and diet in the etiology of breast cancer provided with a -80 degrees C biological bank, we have evaluated the stability of estradiol, free and total testosterone, and prolactin in serum and plasma samples over 3 years of cryoconservation. Study results showed that the subjects maintained almost the same rank by hormonal concentration throughout the 3-year period for all hormones. Looking at the stability over time, estradiol, prolactin, and total testosterone had fairly good performance for both serum and plasma. Serum-free testosterone increased in time up to 30%, whereas progesterone decreased by about 40% of the initial concentration. However, the reliability of the individual categorization by hormonal level suggests the validity of low temperature storage for epidemiological purposes, at least for hormonal parameters.

Measurement of steroid hormones in plasma by isocratic high performance liquid chromatography coupled to radioimmunoassay.

The study of steroidal profiles requires simultaneous determinations of various steroid hormones that cannot be appropriately carried out with the conventional routine immunoassays. Moreover, there are several trials for which the assessment of multiple steroids from a single serum sample is mandatory. In this paper we describe a procedure for simultaneously measuring steroid hormones using a unified solid phase extraction which allows the measurement of both unconjugated and conjugated steroids from 1 ml of sample and a combination of HPLC with isocratic elution followed by RIA. The entire procedure was preliminary carried out for the measurement of testosterone, dehydroepiandrosterone and its sulphated conjugate, androstenedione and 17 hydroxyprogesterone. The use of this technique allows precise and accurate measurements of steroid profile with a single serum aliquot and could be helpful in the diagnosis of various form of endocrine disorders.