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.

Oocyte donation: not all oocyte cryobanks are the same.

RESEARCH QUESTION: Is the efficacy of imported vitrified oocyte donation affected by the cryobank of origin? DESIGN: Longitudinal cohort study, including 249 completed oocyte warming cycles from 200 recipients (January 2016-July 2020). No severe male factor was included. Vitrified oocytes were provided by three Spanish cryobanks. Primary outcome was cumulative live birth delivery rate (CLBR) per completed oocyte warming cycle. RESULTS: After warming 1535 oocytes, 1244 survived (81.0%) and 945 fertilized (76.0%); embryo utilization rate was 65.3%. The overall CLBR per completed cycle was 47.0% but was lower in cryobank 1 (31.2%) versus cryobank 2 (56.0%, P = 0.0010) and cryobank 3 (50.8%, P = 0.0241). Multivariate logistic regression analysis identified survival of four or more oocytes as the strongest predictor for delivery (P = 0.0282). Only 202 out of 249 oocyte warming cycles had four or more survived oocytes in a proportion that was significantly lower in cryobank 1 versus cryobank 2 (70.1% versus 89.0%, P = 0.0020); comparison with cryobank 3 (81.0%) was not significant. In the 202 oocyte warming cycles, CLBR in cryobank 1 (37.0%) was lower versus cryobank 2 (58.8%, P = 0.0115) and cryobank 3 (60.8%, P = 0.0019), suggesting a reduced viability in oocytes from cryobank 1 that survived warming. CONCLUSIONS: Differences were found in the efficacy of imported vitrified oocytes in relation to the cryobank of origin. Each centre needs to evaluate the results internally when starting a collaboration with an oocyte cryobank to establish the necessary measures to maximize treatment efficacy.

Sperm chromosome abnormalities in patients with normal karyotype and in translocation carriers: clinical relevance for assisted reproductive technology.

RESEARCH QUESTION: What is the proportion of chromosomally abnormal spermatozoa in men with a history of reproductive failure, including patients with normal karyotype and carriers of translocations? Should this analysis be included in a clinical setting to define the best treatment options for infertile couples? DESIGN: Aneuploidy for chromosomes XY, 13, 15, 16, 17, 18, 21, 22 was tested by fluorescent in-situ hybridization (FISH) in 1665 samples from couples with normal karyotype having had at least three previous IVF failures, miscarriages, or both (group-A). A FISH test was also carried out in 76 samples from carriers of translocations (group B) to detect the proportion of spermatozoa with unbalanced rearrangement. RESULTS: In group A, the lowest incidence of aneuploid sperm cells was found in men with normozoospermia (1.3%, range 0.09-6.31%) compared with men with moderate oligoasthenoteratozoospermia (2.1%, range 0.41-16.6%, P < 0.001), severe oligoasthenoteratozoospermia (4.7%, range 0.53-30.77, P < 0.001), microepididymal sperm aspiration (3.1%, range 1.19-24.24, P < 0.001) and testicular sperm extraction samples (5.8%, range 1.54-33.3, P < 0.001). In group B, the proportion of spermatozoa with unbalanced rearrangement was significantly higher in reciprocal (63%, range 10.0-87.6%) than in Robertsonian translocations (16%, range 4.3-51.0%, P < 0.001). CONCLUSIONS: Patients with poor prognosis of term pregnancy tend to generate high proportions of chromosomally abnormal spermatozoa, especially in severe male factor cases. Corresponding frequencies occur at wide ranges; therefore, the FISH test is needed to assess the proportion of spermatozoa with altered chromosome condition. A flowchart, which included the FISH test, was designed to assist clinicians guide couples with poor prognosis of pregnancy, on the most indicated treatment options.

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.

Reduction in sperm aneuploidy levels in severe oligoasthenoteratospermic patients after medical therapy: a preliminary report.

The objective of this study was to investigate whether medical therapy can reduce sperm aneuploidy levels and improve the results of intracytoplasmic sperm injection (ICSI) in patients with severe idiopathic oligoasthenoteratospermia (OAT). Thirty-three infertile couples requiring ICSI because of severe idiopathic OAT after at least one unsuccessful ICSI cycle were considered. Semen parameters (concentration, motility and morphology), the percentage of aneuploid sperm and the results of ICSI (the number of oocytes fertilized, embryos transferred, biochemical pregnancies, clinical pregnancies and live births) were compared before and after a 3-month course of treatment with L-carnitine 1 g given twice per day+acetyl-L-carnitine 500 mg given twice per day+one 30-mg cinnoxicam tablet every 4 days. Aneuploidy was assessed using fluorescent in situ hybridisation (FISH) performed on chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22. The results showed that 22 of the 33 patients had a reduced frequency of aneuploid sperm and improved sperm morphology after treatment (group 1), and 11 showed no change (group 2). The numbers of biochemical pregnancies, clinical pregnancies and live births were significantly higher in group 1 than in group 2. No significant difference was found between the groups regarding the numbers of oocytes fertilized and embryos transferred. The side effects were negligible. The numbers of ICSI pregnancies and live births in severe idiopathic OAT patients improved with a course of L-carnitine, acetyl-L-carnitine and cinnoxicam.

DNA integrity is maintained after freeze-drying of human spermatozoa.

OBJECTIVE: To evaluate the effects on human spermatozoa of freeze-drying, also known as lyophilization, and of cryopreservation in liquid nitrogen. DESIGN: Prospective experimental study. SETTING: Reproductive medicine unit and a private IVF center. PATIENT(S): Thirty healthy male donors. INTERVENTION(S): Sperm samples from 30 donors divided as two aliquots, one to be lyophilized and the other to be cryopreserved in liquid nitrogen. MAIN OUTCOME MEASURE(S): Assessment of count, motility, morphology, viability, DNA integrity, chromosomal status, and birefringence properties of lyophilized and cryopreserved human spermatozoa compared with the same parameters in the fresh sample. RESULT(S): Although sperm viability and motility were totally compromised after freeze-drying, the sperm chromatin structure was not altered in comparison with fresh samples, which demonstrated that the procedure did not affect DNA integrity. The sperm-head inner protoplasmic structures were also preserved, which was estimated by assessing the corresponding birefringence characteristics. After cryopreservation with liquid nitrogen, the motility, viability, and DNA integrity of spermatozoa were statistically significantly reduced compared with the fresh samples; the proportion of sperm cells with abnormal head birefringence increased meaningfully. CONCLUSION(S): The process of freeze-drying deeply damages cell membranes; however, unlike with liquid nitrogen preservation, it does not affect DNA integrity.

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.

The number of spermatozoa collected with testicular sperm extraction is a novel predictor of intracytoplasmic sperm injection outcome in non-obstructive azoospermic patients.

The purpose of this study was to determine the relationships between monitors of spermatogenesis and predictors of the intracytoplasmic sperm injection (ICSI) outcome in patients with non-obstructive azoospermia (NOA) undergoing testicular sperm extraction (TESE). Seventy-nine patients with NOA (mean age: 43.6±5.2 years), each of whom yielded (97 000±3040) spermatozoa with conventional TESE, were considered in our analysis. Their partners (mean age: 35.8±5.1 years) underwent a total of 184 ICSI cycles; 632 oocytes were collected, 221 oocytes were injected, 141 oocytes were fertilized, 121 embryos were obtained, 110 embryos were transferred, 14 clinical pregnancies were achieved and only one miscarriage occurred. Multivariate regression analysis indicated relationships between the percentage of fertilized oocytes, transferred embryos and clinical pregnancies with the following variable values: female partner's age, number of spermatozoa collected, testicular volume, male partner's levels of follicle stimulating hormone (FSH), number of oocytes collected, number of oocytes injected and number of ICSI cycles. A significant inverse relationship was found between female partner's age or male partner's FSH levels and biochemical pregnancies. A significant direct relationship emerged between the number of ICSI cycles and the percentage of oocytes fertilized, embryos transferred and biochemical pregnancies, and between the number of spermatozoa collected per testicular biopsy and biochemical pregnancies. The number of spermatozoa was positively linked to the number of clinical pregnancies, independent of the number of ICSI cycles and the number of oocytes collected/injected. The number of spermatozoa collected, FSH level and testicular volume are monitors of spermatogenesis linked to ICSI success.

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.

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.

A study to sustain the hypothesis of the multiple genesis of oligoasthenoteratospermia in human idiopathic infertile males.

Interdependence between sperm concentration, motility, morphology, and the percentage of aneuploid sperm was explored to test whether oligoasthenoteratospermia (OAT) may have a multiple origin in idiopathic infertile males. A total of 174 men (age, 35.8 +/- 4.3 yr) with idiopathic infertility were studied. Seven patients had nonobstructive azoospermia, 55 had severe OAT, 30 had OAT, 27 had isolated alterations of motility, 45 had alterations of morphology and of motility, and 10 had isolated alterations of morphology. The sperm morphology was assessed with strict criteria. The percentage of aneuploid sperm was assessed with fluorescent in situ hybridization for chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22. Relationships between sperm features, and the relationship between sperm features and aneuploidies were analyzed with multivariate regression analysis. Statistical analysis did not find any significant relationship between the percentage of typical forms and sperm concentration or between morphology and motility. On the other hand, a positive and significant relationship was found between sperm concentration and motility. The percentage of aneuploid sperm was inversely and significantly related to the percentage of typical forms but not to motility and concentration. Sperm morphology is an independent characteristic with respect to concentration and motility, whereas it showed a significant inverse relationship with respect to the percentage of aneuploid sperm. This means that idiopathic OAT may occur by means of at least two independent pathways, the first affecting concentration and/or motility and the second affecting morphology.

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.

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.

Glomerular monocyte-macrophage features in ANCA-positive renal vasculitis and cryoglobulinemic nephritis.

Although it is widely known that many macrophages are present in glomeruli of antineutrophil cytoplasmic antibody (ANCA)-positive renal vasculitis (ANCA + RV) and are believed to contribute to necrotizing extracapillary damage, their precise role is not yet completely understood, especially in humans. The goal of this study was to provide evidence of glomerular macrophage properties in human vasculitis. Twenty-five renal biopsies of ANCA + RV and 18 cases of cryoglobulinemic glomerulonephritis (cryoGN), a disease characterized by massive glomerular macrophage infiltration but absence of necrotizing extracapillary lesions, were selected, and macrophage number, adhesion, acute activation, proliferation, and apoptosis were analyzed by immunohistochemistry and in situ hybridization. Accumulation of macrophages in ANCA + RV was found in areas of glomerular active lesions, whereas in cryoGN, they homogeneously occupied the entire glomerular tuft. Considering the areas of accumulation, comparable macrophage numbers were detected in both diseases. Glomerular vascular cell adhesion molecule-1 was found only in ANCA + RV and only in areas of active lesions. Acute macrophage activation (HLA class II, 27E10) and proinflammatory cytokine production (tumor necrosis factor-alpha, interleukin-1alpha) were prominent in ANCA + RV, whereas in cryoGN, 30% of glomerular macrophages seemed activated and cytokine expression was limited to a few glomerular cells (P: = 0.01). Moreover, only in ANCA + RV proliferative markers were shown on glomerular macrophages and apoptotic macrophages were found. From the data, it seems that ANCA + RV and cryoGN differ profoundly in macrophage properties, namely adhesion, proliferation, and apoptotic clearance. Moreover, acute activation and cytokine production seem to be present in a greater number of macrophages in ANCA + RV, giving this disease a stronger severity that could be taken into account for therapeutic strategies.