Failure to detect DNA in blastocoel fluid is associated with a higher live birth rate in both PGT-A and conventional IVF/ICSI cycles.

STUDY QUESTION: Is the presence of DNA in the blastocoel fluid (BF) of expanded blastocysts, assessed by whole genome amplification (WGA), associated with the clinical outcome at the first transfer? SUMMARY ANSWER: At the first transfer, blastocysts with negative BF-WGA have more chance to implant and to develop to term than those with positive BF-WGA results, both in preimplantation genetic testing for aneuploidies (PGT-A) cycles (where only euploid blastocysts resulting from the chromosomal analysis of trophectoderm (TE) biopsies were transferred) and in IVF/ICSI conventional cycles. WHAT IS KNOWN ALREADY: Retrospective studies conducted in patients undergoing PGT-A have shown that the incidence of negative BF-WGA was significantly higher in TE-euploid blastocysts than in TE-aneuploid blastocysts. In addition, after the transfer of TE-euploid blastocysts, the ongoing clinical pregnancy rate was significantly higher in the group with negative BF-WGA compared with those with positive BF-WGA. STUDY DESIGN, SIZE, DURATION: A prospective cohort study including 102 consecutive PGT-A patients (Group 1) and 88 consecutive conventional IVF/ICSI patients (Group 2), was conducted between January 2019 and December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: In both groups, BFs were collected from expanded blastocysts of high grade and processed for WGA. DNA amplification was evaluated by agarose gel electrophoresis for the presence (positive BF-WGA) or absence (negative BF-WGA) of a band. Directly after the BF retrieval, blastocysts from Group 1 underwent TE biopsy and vitrification. In Group 2, blastocysts were vitrified immediately after BF collection. In Group 1, only euploid blastocysts were considered for transfer according to the results of TE biopsies. In both groups, the selection of the blastocyst to be transferred was based on BF-WGA results giving priority, if available, to those with negative amplification. The primary outcome investigated was the live birth rate (LBR) at the first transfer. The main variable under investigation was the negative BF-WGA and results were corrected for confounders (maternal and paternal age, number of retrieved oocytes, male factor) by multiple logistic regression analysis. MAIN RESULTS AND THE ROLE OF CHANCE: In Group 1, 60 patients transferred negative BF-WGA blastocysts and 42 positive BF-WGA blastocysts, and the LBR at the first transfer was 53.3% and 26.2%, respectively (P = 0.0081). After testing for selected confounders in a multiple logistic analysis, the transfer of blastocysts with negative BF-WGA resulted in an odds ratio of (OR) 3.52 (95% CI: 1.48-8.88, P = 0.0057) compared to transfer of positive BF-WGA blastocysts. In Group 2, at the first transfer 30 deliveries resulted from blastocysts with negative BF-WGA (48.4%) and three from the transfer of positive BF-WGA blastocysts in 26 patients (11.5%; P = 0.0014). Multiple logistic analysis indicated that the transfer of blastocysts with negative BF-WGA resulted in an OR 6.89 (95% CI: 1.98-32.95, P = 0.0056) compared to transfer of positive BF-WGA blastocysts. The LBR per transfer and the cumulative LBR per patient showed the same trend. LIMITATIONS, REASONS FOR CAUTION: The study was performed in a single center. WIDER IMPLICATIONS OF THE FINDINGS: The data from this study highlight the heterogeneity of blastocysts of similar morphology, even in those classified as euploid by TE analysis. Failure to detect DNA in BFs after WGA is associated with a significantly higher LBR at the first embryo transfer as well as per transfer and per patient. The processing of the BF by WGA is an easy and cost-effective tool that could become a valuable option to offer patients the highest chances of term pregnancy in the shortest time possible. STUDY FUNDING/COMPETING INTEREST(S): The study received no funding from external sources. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Birefringence properties of human immotile spermatozoa and ICSI outcome.

RESEARCH QUESTION: In sperm samples with complete asthenozoospermia, pregnancies are achieved by intracytoplasmic sperm injection (ICSI), but this condition has a negative impact on fertilization and embryo development owing to the difficulty of identifying viable cells for oocyte injection. Is the selection of sperm cells with head birefringence properties under polarizing light a successful strategy to identify viable spermatozoa? DESIGN: This study included 192 ICSI cycles with complete asthenozoospermia (83 ejaculated and 109 testicular samples) performed under polarized light. Two types of sperm head birefringence were distinguished: partial (presumably reacted spermatozoa) and total (presumably intact acrosome). In some sperm cells, no birefringence was present. The main outcome of the study was the cumulative live birth rate (cLBR) per ICSI cycle. RESULTS: Seventy-three deliveries resulted with 38.0% cLBR per ICSI cycle. The injection of birefringent spermatozoa led to significantly higher rates of fertilization, embryo development and implantation compared with the absence of birefringence (P < 0.001). Similarly, the resulting cLBR were 53.6% and 9.0%, respectively (P < 0.001). Spermatozoa with partial head birefringence yielded significantly higher fertilization and embryo utilization rates compared with total birefringence. The cLBR showed the same trend (62.7% and 46.7%, respectively, P = 0.048). Multiple logistic regression analysis showed the pattern of partial birefringence to be strongly associated with live birth rate. CONCLUSIONS: Immotile sperm cells with birefringence properties under polarized light have higher chances of inducing fertilization and embryo development compared with non-birefringent cells. In addition, a pattern of partial birefringence, associated with a reacted acrosome, is the strongest predictive factor for live birth delivery, both in ejaculated and testicular samples.

Individual participant data meta-analysis of trials comparing frozen versus fresh embryo transfer strategy (INFORM): a protocol.

INTRODUCTION: Existing randomised controlled trials (RCTs) comparing a freeze-all embryo transfer strategy and a fresh embryo transfer strategy have shown conflicting results. A freeze-all or a fresh transfer policy may be preferable for some couples undergoing in-vitro fertilisation (IVF), but it is unclear which couples would benefit most from each policy, how and under which protocols. Therefore, we plan a systematic review and individual participant data meta-analysis of RCTs comparing a freeze-all and a fresh transfer policy. METHODS AND ANALYSIS: We will search electronic databases (Medline, Embase, PsycINFO and CENTRAL) and trial registries (ClinicalTrials.gov and the International Clinical Trials Registry Platform) from their inception to present to identify eligible RCTs. We will also check reference lists of relevant papers. The search was performed on 23 September 2020 and will be updated. We will include RCTs comparing a freeze-all embryo transfer strategy and a fresh embryo transfer strategy in couples undergoing IVF. The primary outcome will be live birth resulting from the first embryo transfer. All outcomes listed in the core outcome set for infertility research will be reported. We will invite the lead investigators of eligible trials to join the Individual participant data meta-analysis of trials comparing frozen versus fresh embryo transfer strategy (INFORM) collaboration and share the deidentified individual participant data (IPD) of their trials. We will harmonise the IPD and perform a two-stage meta-analysis and examine treatment-covariate interactions for important baseline characteristics. ETHICS AND DISSEMINATION: The study ethics have been granted by the Monash University Human Research Ethics Committee (Project ID: 30391). The findings will be disseminated via presentations at international conferences and publication in peer-reviewed journals. PROSPERO REGISTRATION NUMBER: CRD42021296566.

IVF Lite: a smart IVF programme based on mild ovarian stimulation for good prognosis patients.

RESEARCH QUESTION: The IVF Lite programme is based on mild ovarian stimulation including up to three fresh/frozen embryo transfers within 12 months. Is it effective and safe in good prognosis patients? DESIGN: Single-centre prospective study on infertile patients at their first IVF attempt (female age ≤38 years, anti-Müllerian hormone concentrations >1.5 ng/ml and/or FSH ≤10 mIU/ml). Induction of multiple follicular growth was based on a fixed protocol consisting of clomiphene citrate (100 mg/day) from day 3 to 7 of the menstrual cycle and 150 IU of recombinant FSH on days 5, 7 and 9. In case of low follicular recruitment (fewer than four follicles), the cycle was cancelled. The IVF Lite programme was considered complete after a live birth delivery or up to three embryo transfers within 12 months. The primary outcome was the cumulative live birth rate (cLBR) per couples that completed the programme. RESULTS: A total of 369 patients completed the IVF Lite programme, with 239 live births; 132 patients delivered after one embryo transfer (35.8%), 70 after a second embryo transfer (cLBR 54.7%), and 37 after a third attempt (cLBR 64.8%). No cases of ovarian hyperstimulation syndrome or clinical complications occurred. Spontaneous dropout rate from the programme was 4.5%. The cLBR per intention to treat was 46.8%. CONCLUSIONS: The IVF Lite programme proved to be effective and safe in good prognosis patients with a good response to clomiphene citrate stimulation. It was well tolerated and implied low gonadotrophin consumption. Two-thirds of the patients achieved a live birth at the completion of the programme.

Deoxyribonucleic acid detection in blastocoelic fluid: a new predictor of embryo ploidy and viable pregnancy.

OBJECTIVE: To investigate blastocysts, defined as euploid and aneuploid by trophectoderm (TE) cell analysis, for the presence of DNA in the blastocoelic fluid (BF) detected by whole-genomic amplification (WGA); and to correlate the presence of DNA in BF with the clinical outcome after the transfer of TE-euploid blastocysts. DESIGN: Retrospective study. SETTING: In vitro fertilization unit. PATIENT(S): This study included 91 patients performing preimplantation genetic testing for aneuploidy on TE cells from January 2015 to December 2017. In the case of ET, only single blastocyst transfers were performed. INTERVENTION(S): Blastocoelic fluids and TE cells were retrieved from 256 blastocysts before vitrification. All blastocysts were diagnosed by array-comparative genomic hybridization (a-CGH) on TE cells. Amplification and a-CGH of DNA from BFs was performed at a later time after TE biopsy and ET. MAIN OUTCOME MEASURE(S): Whole-genomic amplification of BFs, evaluation of the chromosome condition in BFs and TE cells, and correlation of BF results with the clinical outcome of TE-euploid transferred blastocysts. RESULT(S): The incidence of amplification after WGA was significantly lower in BFs from TE-euploid blastocysts (n = 32, 45%) when compared with the aneuploid ones (n = 150, 81%), resulting in 182 BFs with successful DNA amplification. When submitted to a-CGH, informative results were obtained from 172 BFs. Comparison of these results with those from the corresponding TE cells gave a ploidy concordance of 93.6% and a mean number of aneuploid events per sample that was higher in BFs than in TE cells (2.0 vs. 1.4, respectively). After the transfer of 53 TE-euploid blastocysts, the clinical pregnancy rate was 77% in the group with BF-failed amplification, and 37% after BF-successful amplification. The same trend was found for the ongoing pregnancy rate (68% vs. 31.5%, respectively). CONCLUSION(S): The presence of DNA in BFs detected by WGA is correlated with the blastocyst ploidy condition defined by TE cell biopsy and with the implantation potential of TE-euploid blastocysts. These findings could have a clinical implication for the selection of the most viable embryo for transfer because, after submitting BFs to WGA, priority would be given to TE-euploid blastocysts with BF-failed amplification. Similarly, BF-failed amplification could be an additional selection criterion to prioritize embryos for transfer even in conventional IVF cycles with blastocysts that were vitrified after BF aspiration.

Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid?

OBJECTIVE: To investigate the blastocoelic fluid (BF) for the presence of DNA that could be amplified and analyzed; the extent to which its chromosomal status corresponds to that found in trophectoderm (TE) cells, polar bodies (PBs), or blastomeres; and the identification of segmental abnormalities. DESIGN: Longitudinal cohort study. SETTING: In vitro fertilization unit. PATIENT(S): Fifty-one couples undergoing preimplantation genetic screening or preimplantation genetic diagnosis for translocations by array-comparative genomic hybridization on PBs (n = 21) or blastomeres (n = 30). INTERVENTION(S): BFs and TE cells were retrieved from 116 blastocysts, whose chromosome status had already been established by PB or blastomere assessment. Separate chromosome analysis was performed in 70 BFs. MAIN OUTCOME MEASURE(S): Presence of DNA in BFs, evaluation of the chromosome condition, and comparison with the diagnosis made in TE cells and at earlier stage biopsies. RESULT(S): DNA detection was 82%, with a net improvement after refinement of the procedure. In 97.1% of BFs, the ploidy condition corresponded to that found in TE cells, with one false positive and one false negative. The rate of concordance per single chromosome was 98.4%. Ploidy and chromosome concordance with PBs were 94% and 97.9%, respectively; with blastomeres, the concordances were 95% and 97.7%, respectively. Segmental abnormalities, which were detected in PBs or blastomeres of 16 blastocysts, were also identified in the corresponding BFs. CONCLUSION(S): BF represents to a good extent the blastocyst ploidy condition and chromosome status when compared with TE cells. If the proportion of clinically useful BFs is improved, blastocentesis could become the preferred source of DNA for chromosomal testing.

Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study.

OBJECTIVE: To investigate the presence of DNA in blastocyst fluids (BFs) and to estimate whether the chromosomal status predicted by its analysis corresponds with the ploidy condition in trophectoderm (TE) cells, the whole embryo, and that predicted by polar bodies (PBs) or blastomeres. DESIGN: Prospective study. SETTING: In vitro fertilization unit. PATIENT(S): Seventeen couples undergoing preimplantation genetic screening with the use of array comparative genomic hybridization on PBs (n = 12) or blastomeres (n = 5). INTERVENTION(S): BFs and TE cells were retrieved from 51 blastocysts for separate chromosomal analysis. MAIN OUTCOME MEASURE(S): Presence of DNA in BFs and assessment of the corresponding chromosome condition; correlation with the results in TE cells and those predicted by the analysis done at earlier stages. RESULT(S): DNA was detected in 39 BFs (76.5%). In 38 of 39 cases (97.4%) the ploidy condition of BFs was confirmed in TE cells, and the rate of concordance per single chromosome was 96.6% (904/936). In relation to the whole embryo, the ploidy condition corresponded in all cases with a per-chromosome concordance of 98.1%. The testing of PBs and blastomeres had 93.3% and 100% prediction of BF ploidy condition with a concordance per chromosome of 93.5% and 94%, respectively. CONCLUSION(S): Blastocentesis could represent an alternative source of material for chromosomal testing, because the BF is highly predictive of the embryo ploidy condition and chromosome content. Our data confirm the relevance of the oocyte and of the early-cleavage embryo in determining the ploidy condition of the resulting blastocyst.

Five chromosome segregation in polar bodies and the corresponding oocyte.

For a comprehensive picture of the meiotic process and to follow up its products, five chromosomes were tested by fluorescent in-situ hybridization in both polar bodies (PB) and corresponding 145 oocytes. Results were obtained in 143 sets and the prediction of euploidy or aneuploidy based on PB analysis was confirmed by direct analysis in 140 oocytes (98%). Concordance for all chromosomes was found in 132 oocytes, while in the remaining eight, at least one chromosome did not reflect the prediction made by the corresponding PB. When restricting the analysis to the 132 fully concordant oocytes, 215 errors were found in PB: 58% in PB1 and 42% in PB2. Premature separation of chromatids occurred in 89% of aneuploid PB1, whereas only 11% of errors derived from bivalent non-disjunction. In 19% of meiosis-I errors, a complementary error in meiosis II compensated the error originated in the first meiotic division. In conclusion, the testing of PB predicted reliably the oocyte's chromosome condition. Although limited to five chromosomes, the follow up of meiosis by fluorescent in-situ hybridization provided a full description of chromosome allocation during the two divisions characterizing the nuclear maturation of the oocyte.

Head birefringence properties are associated with acrosome reaction, sperm motility and morphology.

Birefringence in sperm heads reflects an organized and very compacted texture, indicating nuclear and acrosomal structural normality. This study performed a direct analysis of the acrosome integrity in single spermatozoa to verify whether a pattern of total or partial head birefringence reflected the acrosome status. The morphology in fresh samples was assessed according to World Health Organization criteria while the characteristics of birefringence were evaluated by polarized light. Acrosome integrity was evaluated by fluorescein isothiocyanate Pisum sativum agglutinin that binds selectively to the acrosome content. According to the results, a reacted acrosome was present in 96% of spermatozoa with partial birefringence and only in 35% of those with totally birefringent heads. A great proportion of sperm cells with normal morphology showed total birefringence both in the presence (59%) or in the absence of motility (45%; P < 0.01), while in morphologically abnormal spermatozoa the frequency of total birefringence was comparable to that of partial birefringence irrespective of motility (26% and 27%, respectively, in motile spermatozoa; 22% and 19%, respectively, in immotile spermatozoa). These data support a strong association between partial birefringence and reacted acrosome and show that the patterns of birefringence vary depending on sperm motility and morphology.

Prolonged absence of meiotic spindles by birefringence imaging negatively affects normal fertilization and embryo development.

Meiotic spindle (MS) assembly in human oocytes is a dynamic process that can be visualized by computer-assisted microscopy. At extrusion of the first polar body a spindle bridge is detected until the completion of telophase I and its reformation requires approximately 1h. This study analysed 396 oocytes from 112 cycles for fertilization and cleavage according to MS detection at two examinations, 39 and 41 h post-human chorionic gonadotrophin (HCG). All cycles had at least one injected oocyte lacking a visible MS at intracytoplasmic sperm injection (41 h post-HCG). To evaluate the results, oocytes were divided according to the presence (group A) or absence at both observations (group B) of the MS. Compared with group A, group B oocytes had lower normal fertilization rates, higher incidence of three pronuclei and two pronuclei in early dissolution and lower development to blastocyst. Some group A oocytes showed a late MS formation (not visualized at 39 h but at 41 h) and their performance was similar to that of the oocytes with a MS visible at both time points. Although some implantations occurred in group B, these findings suggest that prolonged MS non-detection could be a marker of reduced oocyte competence.

Aneuploidies of chromosomes 1, 4, and 6 are not compatible with human embryos' implantation.

OBJECTIVE: To verify whether chromosomes 1, 4, and 6 have a role in determining oocyte viability. DESIGN: Retrospective study. SETTING: Reproductive Medicine Unit, Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. PATIENT(S): Eighty-five patients with a normal karyotype who had undergone an assisted conception cycle with chromosomal analysis of first polar bodies for chromosomes 13, 15, 16, 18, 21, and 22 (first panel). A clinical pregnancy was obtained in 43 patients, whereas 42 patients were not pregnant. INTERVENTION(S): After conclusion of clinical pregnancies to delivery or abortion, first polar bodies from 85 patients were reanalyzed for chromosomes 1, 4, and 6 (second panel). MAIN OUTCOME MEASURE(S): Aneuploidy frequency, clinical pregnancy outcome. RESULT(S): The aneuploidy rate contributed by chromosome 1, 4, and 6 to the oocytes that were normal for the first panel was significantly higher in the nonpregnant patients (28%) versus the pregnant patients (11%), whereas no difference resulted between term pregnancies (11%) and abortions (10%). This trend was also observed when studying the first polar bodies from the oocytes that originated the transferred embryos. The frequency of aneuploidy for chromosomes 1 and 4 was comparable with that of chromosomes 15, 16, 21, and 22. CONCLUSION(S): Aneuploidy of chromosomes 1, 4, and 6 seems to be related to failed implantation and not to spontaneous abortions.

Impact of oocyte cryopreservation on embryo development.

OBJECTIVE: To verify whether the morphologic evaluation of zygotes and embryos derived from thawed oocytes could provide some relevant information regarding their developmental performance. DESIGN: Fertilization, zygote, and embryo morphology from sibling fresh and frozen oocytes was compared. SETTING: Reproductive Medicine Unit, Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. PATIENT(S): Two hundred thirty-four patients underwent intracytoplasmic sperm injection cycles from which 1,101 spare metaphase II oocytes were cryopreserved. Subsequently, 256 thawing cycles were performed, and 997 oocytes were thawed. INTERVENTION(S): Intracytoplasmic sperm injection was performed on both fresh and frozen oocytes. MAIN OUTCOME MEASURE(S): Fertilization rates, pronuclear zygote morphology, and embryo cleavage rates. RESULT(S): Thawed oocytes had lower chances of being fertilized and developing into top-quality zygotes and regularly cleaving embryos when compared with sibling fresh oocytes irrespective of female age. As a result, the percentage of transferred cycles was significantly lower in frozen cycles compared with fresh cycles (79% and 93%, respectively); the proportion of transferred top-quality embryos followed the same trend. CONCLUSION(S): Reduced fertilization and cleavage rates in frozen cycles when compared with sibling fresh oocytes suggest that, even if surviving thawing, the process of slow freezing has a negative impact on the potential of further growth that is evident as early as the first cleavage divisions.

Birefringence characteristics in sperm heads allow for the selection of reacted spermatozoa for intracytoplasmic sperm injection.

OBJECTIVE: To verify clinical outcome after injection of spermatozoa that have undergone the acrosome reaction (reacted spermatozoa) vs. those still having an intact acrosome (nonreacted spermatozoa). DESIGN: Prospective, randomized study. SETTING: Reproductive Medicine Unit, Italian Society for the Study of Reproductive Medicine, Bologna, Italy. PATIENT(S): According to a prospective randomization including 71 couples with severe male factor infertility, intracytoplasmic sperm injection (ICSI) was performed under polarized light that permitted analysis of the pattern of birefringence in the sperm head. Twenty-three patients had their oocytes injected with reacted spermatozoa, 26 patient's oocytes were injected with nonreacted spermatozoa, and in 22 patients both reacted and nonreacted spermatozoa were injected. INTERVENTION(S): Intracytoplasmic sperm injection was performed under polarized light to selectively inject acrosome-reacted and acrosome-nonreacted spermatozoa. MAIN OUTCOME MEASURE(S): Rates of fertilization, cleavage, pregnancy, implantation, and ongoing implantation. RESULT(S): There was no effect on the fertilizing capacity and embryo development of either type of sperm, whereas the implantation rate was higher in oocytes injected with reacted spermatozoa (39.0%) vs. those injected with nonreacted spermatozoa (8.6%). The implantation rate was 24.4% in the group injected with both reacted and nonreacted spermatozoa. The delivery rate per cycle followed the same trend. CONCLUSION(S): Spermatozoa that have undergone the acrosome reaction seem to be more prone to supporting the development of viable ICSI embryos.

Factors affecting thawed oocyte viability suggest a customized policy of embryo transfer.

OBJECTIVE: To identify factors that might affect the clinical outcome of oocyte slow freezing. DESIGN: Retrospective study. SETTING: Reproductive Medicine Unit, Italian Society for the Study of Reproductive Medicine, Bologna, Italy. PATIENT(S): Patients with spare metaphase II cryopreserved oocytes performing 371 thawing cycles. INTERVENTION(S): Oocytes were cryopreserved by slow freezing<40 hours after hCG administration (group A) and >or=40 hours after hCG administration (group B). Thawed oocytes were inseminated by intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Clinical pregnancy, implantation, abortion, and delivery rates. RESULT(S): Clinical pregnancy rate per thawed cycle (PR) and implantation rate (IR) were significantly higher in group A compared with group B both in young (PR: 25% vs. 9.6%; IR: 18.9% vs. 8.8%) and in older patients (PR: 25% vs. 10.1%; IR: 17.5% vs. 6.7%). In the young patient subgroup, clinical pregnancy and implantation rates with three transferred embryos were higher in group A vs. group B (PR: 72.7% vs. 25%, and IR: 36.4% vs. 12.5%, respectively). This difference was not found in the subgroup of older patients. CONCLUSION(S): The timing at which oocyte cryopreservation is performed and the number of transferred embryos play a key role in the clinical outcome. The suggested cut-off time for cryopreservation is between 39 and 40 hours after hCG administration.

Sperm head's birefringence: a new criterion for sperm selection.

OBJECTIVE: To investigate the characteristics of birefringence in human sperm heads and apply polarization microscopy for sperm selection at intracytoplasmic sperm injection (ICSI). DESIGN: Prospective randomized study. SETTING: Reproductive Medicine Unit, Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. PATIENT(S): A total of 112 male patients had birefringent sperm selected for ICSI (study group). The clinical outcome was compared with that obtained in 119 couples who underwent a conventional ICSI cycle (control group). INTERVENTION(S): The proportion of birefringent spermatozoa was evaluated before and after treatment in relation to the sperm sample quality. Embryo development and clinical outcome in the study group were compared with those in the controls. MAIN OUTCOME MEASURE(S): Proportion of birefringent sperm heads, rates of fertilization, cleavage, pregnancy, implantation, and ongoing implantation. RESULT(S): The proportion of birefringent spermatozoa was significantly higher in normospermic samples when compared with oligoasthenoteratospermic samples with no progressive motility and testicular sperm extraction samples. Although fertilization and cleavage rates did not differ between the study and control groups, in the most severe male factor condition (oligoasthenoteratospermic with no progressive motility and testicular sperm extraction), the rates of clinical pregnancy, ongoing pregnancy, and implantation were significantly higher in the study group versus the controls. CONCLUSION(S): The analysis of birefringence in the sperm head could represent both a diagnostic tool and a novel method for sperm selection.

First meiosis errors in immature oocytes generated by stimulated cycles.

OBJECTIVE: To investigate chromosomal errors detected by first polar body (PB) biopsy in relation to the nuclear maturity of the oocytes. DESIGN: Retrospective study. SETTING: Reproductive medicine unit. PATIENT(S): Eighty-seven cycles were examined by PB biopsy for aneuploidy. Indications were maternal age >or=38 years (49 cycles), repeated IVF failures (22 cycles), and others (16 cycles). INTERVENTION(S): First polar bodies were analyzed for the chromosomes 13, 16, 18, 21, and 22 in both in vivo and in vitro matured oocytes. Euploid oocytes were inseminated by intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Chromosomal status of the analyzed oocytes, development after intracytoplasmic sperm injection, pregnancy, and implantation rates. RESULT(S): In in vitro matured oocytes, the proportion of chromosomal abnormalities was higher than in in vivo matured oocytes (70% vs. 54%, P<.005), with complex abnormalities being the prevailing defect (62% vs. 40%, P<.001). Conversely, the presence of an extra chromatid or the lack of a chromatid was more frequent in in vivo than in in vitro matured oocytes (55% vs. 34%, P<.001). CONCLUSION(S): The low viability of in vitro matured oocytes from stimulated cycles could be related to a significantly higher proportion of chromosomal abnormalities compared with in vivo matured oocytes. Complex abnormalities, involving two or more chromosomes, gave the strongest contribution to the detected increase.

The beneficial effects of preimplantation genetic diagnosis for aneuploidy support extensive clinical application.

The aim of this study was to evaluate the clinical impact of preimplantation genetic diagnosis (PGD) for aneuploidy on 193 patients who subsequently achieved 208 clinical pregnancies, in relation to their reproductive history. The 208 clinical pregnancies included in the study resulted from 1029 assisted conception cycles in combination with PGD for aneuploidy in 740 couples with a history of poor reproductive performance. According to the reproductive history of the 193 patients, 61 had previously experienced 112 pregnancies with 105 abortions and seven deliveries, corresponding to 3.6% take-home baby rate and 10.9% implantation rate. During the PGD cycle, preimplantation embryos were analysed for 5-9 chromosomes. The transfer of euploid embryos was performed in 699 cycles (68% of oocyte retrievals), generating 171 term pregnancies with 210 infants born, whereas 34 aborted spontaneously and three were ectopic, giving a take-home baby rate per pregnant patient of 88.6% and an ongoing implantation rate per pregnant patient of 53.2%. According to these data, selection made in preimplantation embryos against chromosomal abnormalities is associated with a significantly higher (P < 0.001) take-home baby rate when compared with the previous reproductive history of the parents.

Frequency of aneuploidy in sperm from patients with extremely severe male factor infertility.

BACKGROUND: A protocol for the chromosomal analysis of sperm samples with a severely reduced number of sperm cells was designed. METHODS: A severe male factor condition was the main cause of infertility for 38 couples: 27 were oligoasthenoteratospermic (OAT) and 11 with non-obstructive azoospermia underwent testicular sperm extraction (TESE). A two-round fluorescence in situ hybridization (FISH) protocol was performed with probes specific for the chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22. The recording of the position of each sperm cell at the microscope allowed diagnosis of each spermatozoon for the nine tested chromosomes. RESULTS: A mean number of 122+/-78.5 sperm were diagnosed per patient with an incidence of total abnormalities corresponding to 13.4%. chi2-tests for the observed frequencies and goodness-of-fit test were highly significant in all cases. A significantly higher proportion of total aneuploidy was detected in 79% of the tested samples compared to the normal population. Testicular sperm were significantly more prone to aneuploidy than ejaculated sperm. CONCLUSIONS: The designed FISH protocol for the analysis of severe OAT and TESE sperm samples is reliable, implying that the studied sample is representative of the original population. In view of the high incidence of aneuploidy in most severe OAT and TESE sperm, the FISH analysis of pathological sperm samples can be routinely performed in order to estimate the chances of the paternal contribution to aneuploidy in the resulting embryos.

Sperm and blastomere aneuploidy detection in reproductive genetics and medicine.

The use of multiple probes in fluorescence in situ hybridization (FISH) permits the simultaneous analysis of several chromosomes in both blastomeres and spermatozoa. Preimplantation genetic diagnosis (PGD) for aneuploidy provides information on embryonic chromosomal status, enabling the selection of embryos carrying aneuploid condition. This strategy directly affects implantation, as documented for patients with a poor prognosis for pregnancy, who have the tendency to generate high proportions of chromosomally abnormal embryos. PGD for aneuploidy also has contributed information on early phases in human embryology by clarifying the molecular basis in some cases of irregular development. Multicolor FISH has also been used to study chromosomes on spermatozoa. Experimental strategies and modifications enabled the analysis of samples with a very low number of sperm cells, including samples retrieved from the genital tract or directly from the testicular tissue. The results confirmed that the incidence of aneuploidy increases proportionally with the severity of the male-factor condition. This observation suggests that, in selected cases, the paternal contribution to aneuploidy in the developing conceptus could be more relevant than expected from general data from aborted fetuses and live births.

The combination of polar body and embryo biopsy does not affect embryo viability.

BACKGROUND: The biopsy of both polar bodies and a blastomere from the same embryo was investigated as an approach aimed at increasing the quantity of DNA available for genetic analysis in preimplantation embryos. METHODS: In 113 cycles, preimplantation genetic diagnosis (PGD) was performed for aneuploidy: 19 cycles underwent polar body biopsy, 32 cycles had both polar body and blastomere biopsy done, and the remaining 62 cycles underwent blastomere biopsy. The chromosomal analysis was performed in a two-round fluorescence in situ hybridization (FISH) protocol with probes specific for the chromosomes X, Y, 13, 15, 16, 18, 21 and 22. RESULTS: The morphological evaluation of the analysed embryos demonstrated similar rates of development irrespective of the biopsy procedure. Accordingly, the implantation rate did not differ significantly in the three biopsy groups and was 15% after polar body biopsy, 26% after the combined biopsy procedures of polar bodies and blastomeres, and 25% after blastomere biopsy. CONCLUSIONS: The removal of a blastomere subsequent to polar body biopsy does not seem to have negative effects on embryo viability. This approach could be especially valuable for a combined diagnosis of aneuploidy and single-gene disorders in preimplantation embryos generated by couples at high reproductive risk.