Impact of cell loss after warming of human vitrified day 3 embryos on obstetric outcome in single frozen embryo transfers.

PURPOSE: Does cell loss (CL) after vitrification and warming (V/W) of day 3 embryos have an impact on live birth rate (LBR) and neonatal outcomes? METHOD: This retrospective analysis includes cleavage stage day 3 embryos vitrified/warmed between 2011 and 2018. Only single vitrified/warmed embryo transfers were included. Pre-implantation genetic screening, oocyte donation, and age banking were excluded from the analysis. The sample was divided into two groups: group A (intact embryo after warming) and group B (≤ 50% blastomere loss after warming). RESULTS: On the total embryos (n = 2327), 1953 were fully intact (83.9%, group A) and 374 presented cell damage (16.1%, group B). In group B, 62% (232/374) of the embryos had lost only one cell. Age at cryopreservation, cause of infertility, insemination procedure, and semen origin were comparable between the two groups. The positive hCG rate (30% and 24.3%, respectively, for intact vs CL group, p = 0.028) and LBR (13.7% and 9.4%, respectively, for intact vs CL group, p = 0.023) per warming cycle were significantly higher for intact embryos. However, LBR per positive hCG was equivalent between intact and damaged embryos (45.6% vs 38.5%, respectively, p = 0.2). Newborn measurements (length, weight, and head circumference at birth) were comparable between the two groups. Multivariate logistic regression showed that the presence of CL is not predictive for LB when adjusting for patients' age. CONCLUSIONS: LBR is significantly higher after transfer of an intact embryo compared to an embryo with CL after warming; however, neonatal outcomes are comparable between the two groups.

Thyroid hormones T3 and T4 regulate human luteinized granulosa cells, counteracting apoptosis and promoting cell survival.

PURPOSE: Fine and balanced regulation of cell proliferation and apoptosis are key to achieve ovarian follicle development from the primordial to the preovulatory stage and therefore assure female reproductive function. While gonadotropins are the major and most recognized regulators of follicle cell growth and function, other factors, both systemic and local, play equally important roles. This work is aimed at evaluating the effects of thyroid hormones (THs) on human granulosa luteinized (hGL) viability. METHODS: Human GL cells derived from assisted reproduction treatments were exposed to T3 or T4. Cell viability was evaluated by MTT assay. Apoptosis was evaluated by the TUNEL assay and active caspase-3 staining. StAR, CYP19A1,Caspase-3, P53 and BAX mRNA were evaluated by real-time PCR. LC3-I/-II, AKT and pAKT were evaluated by western blot. RESULTS: T3 and T4 promoted cell viability in a dose-dependent modality and modulate StAR and CYP19A1 expression. T3 and to a lesser extent T4 mitigated cell death induced by serum starvation by inhibition of caspase-3 activity and expression of P53 and BAX; and attenuate cell death experimentally induced by C2-ceramide. Cell death derived from starvation appeared to be involved in autophagic processes, as the levels of autophagic markers (LC3-II/LC3-I ratio) decreased when starved cells were exposed to T3 and T4. This effect was associated with an increase in pAkt levels. CONCLUSION: From the present study, THs emerge as potent anti-apoptotic agents in hGL cells. This effect is achieved by inhibiting the apoptosis signalling pathway of BAX and caspase-3, while maintaining active the PI3K/AKT pathway.

Pituitary block with gonadotrophin-releasing hormone antagonist during intrauterine insemination cycles: a systematic review and meta-analysis of randomised controlled trials.

BACKGROUND: Several randomised controlled trials (RCTs) have investigated the usefulness of pituitary block with gonadotrophin-releasing hormone (GnRH) antagonists during intrauterine insemination (IUI) cycles, with conflicting results. OBJECTIVE: The aim of the present systematic review and meta-analysis of RCTs was to evaluate the effectiveness of GnRH antagonist administration as an intervention to improve the success of IUI cycles. SEARCH STRATEGY: Electronic databases (MEDLINE, Scopus, EMBASE, Sciencedirect) and clinical registers were searched from their inception until October 2017. SELECTION CRITERIA: Randomised controlled trials of infertile women undergoing one or more IUI stimulated cycles with GnRH antagonists compared with a control group. DATA COLLECTION AND ANALYSIS: The primary outcomes were ongoing pregnancy/live birth rate (OPR/LBR) and clinical pregnancy rate (CPR). Pooled results were expressed as odds ratio (OR) or mean differences with 95% confidence interval (95% CI). Sources of heterogeneity were investigated through sensitivity and subgroups analysis. The body of evidence was rated using GRADE methodology. Publication bias was assessed with funnel plot, Begg's and Egger's tests. MAIN RESULTS: Fifteen RCTs were included (3253 IUI cycles, 2345 participants). No differences in OPR/LBR (OR 1.14, 95% CI 0.82-1.57, P = 0.44) and CPR (OR 1.28, 95% CI 0.97-1.69, P = 0.08) were found. Sensitivity and subgroup analyses did not provide statistical changes in pooled results. The body of evidence was rated as low (GRADE 2/4). No publication bias was detected. CONCLUSION: Pituitary block with GnRH antagonists does not improve OPR/LBR and CPR in women undergoing IUI cycles. TWEETABLE ABSTRACT: Pituitary block with GnRH antagonists does not improve the success of IUI cycles.

Correction to: Contribution of cryopreservation to the cumulative live birth rate: a large multicentric cycle-based data analysis from the Italian National Registry.

The original article unfortunately contained a mistake. The names of the collaborators were captured as authors of the article.

Contribution of cryopreservation to the cumulative live birth rate: a large multicentric cycle-based data analysis from the Italian National Registry.

PURPOSE: To estimate the contribution of cryopreservation to the cumulative live birth rate (CLBR) after law modification in Italy in the era of vitrification and freeze-all. METHODS: The Italian National Registry performed a cycle-based data collection. Nine Italian IVF clinics were involved incorporating a total of 10,260 fresh cycles performed between January 2015 and April 2016 resulting in 9273 oocyte retrievals and 3266 subsequent warming cycles from the same oocyte retrievals performed up to December 2016. Mean female age was 37 ± 4.3 years. Primary outcome measure was CLBR per oocyte retrieval. Confounding factors were tested in multivariate regression analysis, and the relative impact of cryopreservation to the CLBR in different patient categories was calculated. RESULTS: CLBR per oocyte retrieval was 32.6%, 26.5%, 18.7%, 13.0%, and 5.5% for women younger than 36, aged 36-39, 40-41, and older than 41 years, respectively. The total relative contribution of oocyte/embryo cryopreservation was 40.6% (95% CI 38.41-42.75). An association between maternal age, number of oocytes retrieved, fertilization rate, cryopreservation, and cumulative live birth was shown. When adjusted for confounders, a 2.3-fold increase was observed in the chance of live birth when cryopreservation was performed (OR 2.3; 95% CI 1.99-2.56). In high responder patients (> 15 oocytes retrieved) where freeze-all was applied in 67.6% of cycles to avoid the risk of hyper stimulation syndrome, the relative contribution of vitrification to the CLBR was 80.6%. CONCLUSIONS: Cryopreservation is essential in IVF and should always be available to patients to optimize success rates. Multicentric, cycle-based data analyses are crucial to provide infertile couples, clinicians, and regulatory bodies with accurate information on IVF effectiveness including fresh and cryopreserved cycles.

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.

Randomized, controlled, open-label, non-inferiority study of the CONSORT algorithm for individualized dosing of follitropin alfa.

In this randomized, controlled, open-label, phase IV study, ovarian response after a follitropin alfa starting dose determined by the CONSORT calculator was compared with a standard dose (150 IU). Normo-ovulatory women (aged 18-34 years) eligible for assisted reproductive techniques were recruited (23 centres: nine European countries and Chile); 200 women were randomized (CONSORT [n = 96]; standard dosing [n = 104]). Significantly lower mean daily (121.5 versus 167.4 IU; P < 0.001) and total (1288.5 versus 1810.0 IU; P < 0.001) doses of follitropin alfa were administered in the CONSORT group. Clinical pregnancy rates were CONSORT (36.0%) and standard dosing (35.5%); estimated difference (confidence interval 0.6%; -13.5 to 14.6). Ovarian hyperstimulation syndrome occurred in 6.3% and 12.5% of patients in the CONSORT and standard-dosing groups, respectively. The primary efficacy analysis found a significantly lower mean [SD] number of oocytes retrieved in the CONSORT (10.0 [5.6]; P = 0.037) versus standard-dosing group (11.8 [5.3]). Although the CONSORT calculator was statistically inferior to standard dosing in the number of oocytes retrieved, clinical pregnancy rates (fresh embryo transfers) were similar in both groups, and incidence of ovarian hyperstimulation syndrome was lower in the CONSORT group.

Vibrational characterization of female gametes: a comparative study.

In the last few years, vibrational spectroscopies have been widely applied in biology and medicine, as a synergic support to commonly used analytical and diagnostic techniques. This review summarizes the relevant researches carried out by using FTIR and Raman spectroscopy on oviparous and mammalian gametes, including human ones.

Individualizing FSH dose for assisted reproduction using a novel algorithm: the CONSORT study.

The CONSORT dosing algorithm individualizes recombinant human FSH (r-hFSH) doses for assisted reproduction technologies, assigning 37.5 IU increments according to patient characteristics: basal FSH, body mass index, age and antral follicle count. A prospective, uncontrolled, international, 18-centre, pilot study of normo-ovulatory women aged 18-34 years inclusive undergoing a long agonist treatment protocol was performed. Follitropin alfa filled-by-mass (GONAL-f®) dose was assigned by the algorithm and was intended to be altered only for risk of ovarian hyperstimulation syndrome (OHSS). Primary end-point was number of oocytes retrieved. Dose groups containing ≥5 patients were analysed: 75 IU (n = 48), 112.5 IU in = 45), 150 IU (n = 34), 187.5 IU (n = 24), 225 IU (n = 10). Cancellations due to inadequate response were higher than expected in the 75 IU group (12/48). Overall, a median of 9.0 oocytes were retrieved (8.5, 8.0, 10.0, 12.0 and 8.0 in the 75, 112.5, 150, 187.5 and 225 IU groups respectively). Clinical pregnancy rates/cycle started were 31.3, 31.1, 35.3, 50.0 and 20.0%, respectively (overall, 34.2%). Two patients had severe OHSS. Use of the CONSORT algorithm achieved an adequate oocyte yield and good pregnancy rates in this preliminary study. Adjustment of the algorithm could reduce cancellation rates.

Transcriptomic profiling of human oocytes: association of meiotic aneuploidy and altered oocyte gene expression.

The ability to identify oocytes with the greatest potential for producing a viable embryo would be of great benefit to assisted reproductive treatments. One of the most important defects affecting oocytes is aneuploidy. Aneuploidy is also closely related with advancing maternal age, a phenomenon not well understood. This study combined a comprehensive cytogenetic investigation of 21 oocytes with a detailed assessment of their transcriptome. The first polar body was removed from all oocytes and aneuploidy assessed using comparative genomic hybridization. Preliminary mRNA transcript data were produced with the use of microarrays for seven of the corresponding oocytes (three normal and four aneuploid). The results obtained for normal and aneuploid oocytes were compared and 327 genes were found to display statistically (P < 0.05) significant differences in transcript levels. Ninety-six of these genes were further assessed in seven aneuploid and seven normal oocytes using real-time PCR. The results indicated that aneuploidy is associated with altered transcript levels affecting a subset of genes. A link between mRNA transcript numbers and age was also observed. The possibility that different transcript levels in the oocyte have an impact on cellular pathways remains to be proven. However, it may be significant that some of the highlighted genes produce proteins involved in spindle assembly and chromosome alignment. Additionally, several genes with altered amounts of transcript produce cell surface or excretory molecules, and could potentially serve as targets for non-invasive oocyte aneuploidy assessment.

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.

Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies.

Over the past decade, the number of reported live births resulting from oocyte cryopreservation has rapidly increased. To appreciate the true number of children born, verified live births were tabulated and assessed. A literature search was performed; authors were then contacted to verify birth outcomes and provide updates. A database including all verified live born infants was constructed. A total of 58 reports (1986-2008) were reviewed, which included 609 live born babies (308 from slow freezing, 289 from vitrification and 12 from both methods). Additionally, 327 other live births were verified. Of the total 936 live borns, 1.3% (12) were noted to have birth anomalies: three ventricular septal defects, one choanal and one biliary atresia, one Rubinstein-Taybi syndrome, one Arnold-Chiari syndrome, one cleft palate, three clubfoot and one skin haemangioma. Compared with congenital anomalies occurring in naturally conceived infants, no difference was noted. With more live born data accumulating, this procedure may become mainstream as a fertility preservation option, particularly for women diagnosed with malignancy requiring cytotoxic therapy. A registry would help to assure the safest, most expeditious development of this technology.

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.

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.

Individualizing FSH dose for assisted reproduction using a novel algorithm: the CONSORT study.

The CONSORT dosing algorithm individualizes recombinant human FSH (r-hFSH) doses for assisted reproduction technologies, assigning 37.5 IU increments according to patient characteristics: basal FSH, body mass index, age and antral follicle count. A prospective, uncontrolled, international, 18-centre, pilot study of normo-ovulatory women aged 18-34 years inclusive undergoing a long agonist treatment protocol was performed. Follitropin alpha filled-by-mass (GONAL-f) dose was assigned by the algorithm and was intended to be altered only for risk of ovarian hyperstimulation syndrome (OHSS). Primary end-point was number of oocytes retrieved. Dose groups containing >or=5 patients were analysed: 75 IU (n = 48), 112.5 IU (n = 45), 150 IU (n = 34), 187.5 IU (n = 24), 225 IU (n = 10). Cancellations due to inadequate response were higher than expected in the 75 IU group (12/48). Overall, a median of 9.0 oocytes were retrieved (8.5, 8.0, 10.0, 12.0 and 8.0 in the 75, 112.5, 150, 187.5 and 225 IU groups respectively). Clinical pregnancy rates/cycle started were 31.3, 31.1, 35.3, 50.0 and 20.0%, respectively (overall, 34.2%). Two patients had severe OHSS. Use of the CONSORT algorithm achieved an adequate oocyte yield and good pregnancy rates in this preliminary study. Adjustment of the algorithm could reduce cancellation rates.

Sperm selection with density gradient centrifugation and swim up: effect on DNA fragmentation in viable spermatozoa.

Subjects increasing sperm DNA fragmentation (sDF) during Density Gradient Centrifugation (DGC), a common sperm selection procedure in Assisted Reproduction Techniques (ARTs), experience a 50% lower probability of pregnancy. Hence, identification of these subjects is of clinical importance. Here, we investigated whether such subjects are identified with higher accuracy detecting DNA fragmentation in viable (viable sDF) instead of total spermatozoa (total sDF) and whether swim up, an alternative procedure to DGC, does not increase sDF. With DGC, we identified 10/20 subjects increasing total sDF, and 2 more subjects using viable sDF. With swim up, we identified 8/40 subjects increasing total sDF, and 8 more subjects using viable sDF. In addition, viable sDF reveals more accurately the increase of the damage when it occurs. Finally, a multivariate analysis demonstrated that the proportional increase of sDF was higher after DGC respect to swim up. In conclusion, viable sDF is a more accurate parameter to reveal the increase of the damage by selection both with swim up and DGC. Swim up increases sDF in some samples, although at a lesser extent than DGC, suggesting that it should be used to select spermatozoa for ARTs when possible.

Focus on breast and ovarian cancer.

In reproductive medicine the widespread use of ovarian stimulation has focused interest on the possible relation between induction of ovulation and breast and ovarian cancer. The epidemiological studies published so far are reassuring but not devoid of methodological problems, such as small populations, short follow-ups, and lack of information on confounding factors like oral contraceptive use. In younger patients to preserve fertility before radio- or chemotherapy, the American Society of Clinical Oncology recommended embryo cryopreservation as an established procedure. Moreover, experimental procedure as oocytes cryopreservation, ovarian suppression with GnRH analogues and cortical ovarian tissue freezing probably will be routinely used in the future. A reasonable proposal to protect patients from the risk of estrogen peaks in the case of estrogen receptor positive (ER+) breast cancer, ovarian stimulation with tamoxifen or letrozole could be right. Powered studies are necessary to control the safety and effectiveness of the different fertility preservation options. In this review we evaluated the relation between induction of ovulation and breast and ovarian cancer and then the possible options for fertility preservation in patients with these types of tumors.

Pelvic factor infertility: diagnosis and prognosis of various procedures.

Infertile women are examined to exclude tubal occlusion or a pelvic factor through indirect tests, such as hysterosalpingography (HSG), sonohysterosalpingography/hysterosalpingosonography (SH), and/or laparoscopy (Lps). Assisted reproductive technologies (ART) are proposed to resolve infertility according to the results of the above-mentioned diagnostic procedures. Today, Lps still represents the second option after several failures of in vivo attempts and before moving to conceive in vitro. The aim of this study was to establish the diagnostic power of HSG and SH compared with that of Lps and the efficacy of ART when each single test is used as an inclusion criterion. We recruited 2560 infertile women (aged 20 to 35) to undergo diagnostic and therapeutic procedures to address their infertility in our clinical theatre. Of these, 1080 women underwent Lps and hysteroscopy (Group 1), 525 underwent HSG (Group 2), and 955 underwent SH (Group 3). The positive and negative predictive values of sonosalpingosonography were 75.8% and 91.2% and those of hysterosalpingography were 71.8% and 88.2%, respectively. Endometriosis (stage II-IV of the revised American Society for Reproductive Medicine [ASRM] classification) was diagnosed laparoscopically in 344 out of 1080 women (32%). Only 44 women (13%) with endometriosis showed bilateral tubal occlusion. Pelvic factors other than tubal occlusions are neither diagnosed nor treated in a timely manner by indirect tubal patency tests. The conventional use of HSG and/or SH may increase the time required to find an adequate treatment by which to achieve a successful pregnancy.

Ultrastructure of human mature oocytes after slow cooling cryopreservation with ethylene glycol.

The morphological characteristics of frozen-thawed human mature oocytes (n = 12) were studied by light and transmission electron microscopy following cryopreservation using a slow cooling protocol including increasing concentrations of ethylene glycol (0.5-1.5 mol/l) and sucrose 0.2 mol/l in the freezing solution. Fresh human mature oocytes (n = 12) were used as controls. Fresh and frozen-thawed oocytes appeared rounded in section, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Disorganization of mitochondria-smooth endoplasmic reticulum aggregates and a decreased complement of microvilli and cortical granules were frequently observable in frozen-thawed oocytes. Increased density of the inner zona pellucida, possibly related to the occurrence of zona 'hardening', was sometimes found associated with a reduced amount of cortical granules. In addition, delamination of the zona pellucida was evident in some frozen-thawed samples. Finally, numerous vacuoles and secondary lysosomes were detected in the ooplasm of most frozen-thawed oocytes. In conclusion, frozen-thawed oocytes treated with ethylene glycol may show a variety of ultrastructural alterations, possibly related, at least in part, to the use of this cryoprotectant. Thus, the ethylene glycol-based protocol of slow cooling herein described does not seem to offer significant advantages in terms of oocyte structural preservation.