The heparin-glutathione test: an alternative to the hypo-osmotic swelling test to select viable sperm for intracytoplasmic sperm injection

Abstract Objective: To evaluate the heparin-glutathione test (HEGLUT) for the selection of viable sperm for intracytoplasmic sperm injection (ICSI). Design: A prospective study. Setting: Department of Pediatrics, Obstetrics and Gynecology, University of Valencia and Instituto Valenciano de Infertilidad. Patient(s): Semen samples from healthy donors and patients with infertility. Intervention(s): Sperm samples were kept in culture for different periods in Ham's F-10 medium supplemented or not supplemented with heparin, reduced glutathione (GSH), or a heparin-GSH mixture. Control and heparin-GSH–treated spermatozoa were injected into hamster oocytes. The HEGLUT and ICSI were performed. Main O…

INCREASED CONCORDANCE RATES BETWEEN EMBRYO CELL-FREE DNA AND TROPHECTODERM BIOPSIES ARE RELATED TO FEMALE AGE INDEPENDENTLY OF CULTURE CONDITIONS

Clinical application of embryo aneuploidy testing by next-generation sequencing

Abstract We review here the evolution in the field of embryo aneuploidy testing over the last 20 years, from the analysis of a subset of chromosomes by fluorescence in situ hybridisation to the transition toward a more comprehensive analysis of all 24 chromosomes. This current comprehensive aneuploidy testing most commonly employs next-generation sequencing (NGS). We present our experience in over 130 000 embryo biopsies using this technology. The incidence of aneuploidy was lower in trophectoderm biopsies compared to cleavage-stage biopsies. We also confirmed by NGS that embryo aneuploidy rates increased with increasing maternal age, mostly attributable to an increase in complex aneuploid …

How many single embryo transfers would be needed to perform an euploid embryo transfer according to the age of the patient?

Embryo Aneuploidy Screening for Unexplained Recurrent Miscarriage: A Minireview

Problem:  The aim of this study was to investigate the incidence of chromosomal abnormalities in unexplained recurrent miscarriage (RM) patients and assess the role of pre-implantation genetic diagnosis (PGD) in preventing subsequent pregnancy loss and improving pregnancy outcome. Method of Study:  Pre-implantation genetic diagnosis was performed in 241 RM cycles and in 35 cycles in patients undergoing PGD for sex-linked diseases (control group). Chromosomes 13, 16, 18, 21, 22, X and Y were analysed by fluorescence in situ hybridization. Results:  The implantation and pregnancy rates in RM patients were 26.4 and 36.5% versus 20.6 and 29.0% in the control group, respectively. The percentage …

Analysis of chromosomal abnormalities in testicular and epididymal spermatozoa from azoospermic ICSI patients by fluorescence in-situ hybridization

BACKGROUND: An increased incidence of numerical chromosomal abnormalities has been reported in the ejaculated spermatozoa of infertile patients. However, there are few cytogenetic studies of testicular and epididymal spermatozoa, and their results are still controversial. METHODS: Fluorescence in-situ hybridization (FISH) analysis of chromosomes 13, 18, 21, X and Y was performed on seven testicular samples and two epididymal samples from patients with obstructive azoospermia (OA), and on 13 testicular samples from patients with non-obstructive azoospermia (NOA). Five ejaculated sperm samples from normozoospermic fertile donors were evaluated as a control group. RESULTS: Both epididymal sper…

Bisphenol A Effects on the Growing Mouse Oocyte Are Influenced by Diet1

Growing evidence suggests that exposure to bisphenol A (BPA) has the ability to disrupt several different stages of oocyte development. To date, most attention has focused on the effects of BPA on the periovulatory oocyte, and considerable variation is evident in the results of these studies. In our own laboratory, variation in the results of BPA studies conducted at different times appeared to correlate with changes in mill dates of animal feed. This observation, coupled with reports by others that dietary estrogens in feed are a confounding variable in studies of endocrine-disrupting chemicals, prompted us to evaluate the effect of diet on the results of BPA studies of the periovulatory o…

Preimplantation genetic diagnosis by fluorescence in situ hybridization: clinical possibilities and pitfalls.

Preimplantation genetic diagnosis using the fluorescence in situ hybridization technique (FISH) is being used widely to prevent the transmission of sex-linked diseases, to screen for translocations, and for aneuploidy screenng in specific invitro fertilization (IVF) patient groups, along with FISH analysis of spematozoa in intertile men. In this study, we aim to critically analyze our clinical results in patients at risk of transmitting sex-linked diseases (n = 55), in carriers of translocations (n = 43), in women who have recurent miscarriage (two or more miscarriages) (n = 128), recurrent IVF failure (three or more failed IVF attempts) (n = 47), and patients of advanced maternal age (37 y…

Epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole for six Candida species as determined by the colorimetric Sensititre YeastOne method

ABSTRACT In the absence of clinical breakpoints (CBP), epidemiological cutoff values (ECVs) are useful to separate wild-type (WT) isolates (without mechanisms of resistance) from non-WT isolates (those that can harbor some resistance mechanisms), which is the goal of susceptibility tests. Sensititre YeastOne (SYO) is a widely used method to determine susceptibility of Candida spp. to antifungal agents. The CLSI CBP have been established, but not for the SYO method. The ECVs for four azoles, obtained using MIC distributions determined by the SYO method, were calculated via five methods (three statistical methods and based on the MIC 50 and modal MIC). Respectively, the median ECVs (in mg/lit…

FISH screening of aneuploidies in preimplantation embryos to improve IVF outcome

Preimplantation genetic diagnosis (PGD) has transformed the approach to the infertility patient in the IVF setting. Although the principal applications of PGD have been to prevent the transmission of sex-linked diseases, in time and with growing knowledge of the chromosomal abnormalities observed in preimplantation embryos, its applications have widened. Nowadays, apart from its implications in the prevention of transmission of chromosomal and genetic abnormalities, PGD is being used with increased frequency to improve the IVF outcome in patients with advanced maternal age (or =38 years of age), recurrent miscarriage (or =2 miscarriages), recurrent IVF failure (or =3 failed IVF attempts) an…

False positive rate of an arrayCGH platform for single-cell preimplantation genetic screening and subsequent clinical application on day-3

In this work, false positive rate of an arrayCGH platform for its use in day-3 single-blastomere analysis was calculated. For this purpose, 38 embryos diagnosed as abnormal on day-3 by FISH were re-biopsied on day-4. Single-cell day-4 arrayCGH diagnosis was then performed. A successful amplification was obtained in 97.4 % (37/38) of the day-4 cells analysed by arrayCGH. Day-3 FISH and day-4 arrayCGH diagnosis were concordant in 35/37 cases. The two discordant embryos were spread and all the cells from each embryo were re-analysed by FISH on day 5. The same error rate (2.7 %) for day-3 FISH and day-4 arrayCGH was obtained when comparing day-5 FISH re-analysis. After this pre-clinical phase, …

Impact of chromosomal abnormalities on preimplantation embryo development.

Objectives To evaluate the influence of numerical chromosomal abnormalities on preimplantation embryo development. Methods This study includes 6936 embryos from 1245 women undergoing preimplantation genetic diagnosis (PGD). Indications for aneuploidy screening were: recurrent miscarriages, implantation failure, severe male factor, advanced maternal age, and mixed causes. Embryo biopsy was performed on day 3, and embryos were co-cultured until day 5, when embryo transfer was performed. Results In the aneuploidy screening regimen, normal euploid embryos showed significantly higher blastocyst rates (68.2%) compared to chromosomally abnormal (42.8%, p < 0.0001) and mosaic (53.7%, p < 0.0001) em…

New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization

The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n=203); repetitive implantation failure (n=188); severe male factor (n=116); previous trisomic pregnancy (n=33); and advanced maternal age (n=880). CCS was performed in cycles with fresh oocytes and embryos (n=774); mixed cycles with fresh and vitrified oocytes (n=320); mixed cycles with fresh and vitrified day-2 embryos (n=235); and mixed cycles with fresh and vitrified day-3 embryos (n=91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo tran…

The role of thrombophilia and thyroid autoimmunity in unexplained infertility, implantation failure and recurrent spontaneous abortion.

The role of thrombophilia and thyroid autoimmunity in unexplained infertility (UI), implantation failure (IF) and recurrent spontaneous abortion (RSA) is controversial and poorly understood.From March, 2004 to January, 2007, 119 women were prospectively included: 32 oocyte donors, 31 patients with UI, 26 with IF and 30 with RSA. The IF and RSA groups presented normal preimplantation genetic screening. Protein C, protein S, antithrombin III, lupus anticoagulant, activated protein C resistance (APCR), immunoglobulin M and G anticardiolipin antibodies, homocystine, Factor V Leiden, prothrombin G20210A mutation, methylentetrahydrofolate reductase C677T mutation, thyroid-stimulating hormone (TSH…

Fertilization after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa.

Objective To assess the possibility of cryopreserving testicular tissue extracted sperm for intracytoplasmic sperm injection (ICSI). Design A report of two cases. Our study was approved by the Ethical Committee at the Instituto Valenciano de Infertilidad. Setting In vitro fertilization program at the Instituto Valenciano de Infertilidad. Patients Two azoospermic patients with severe spermatogenic failure but with focal spermatogenesis on testicular biopsies. In both cases, a first ICSI attempt with fresh testicular biopsy extracted sperm was unsuccessful. Interventions Cryopreservation of testicular spermatozoa in 100-µL "pills." Intracytoplasmic sperm injection with thawed testicular sperm…

The why, the how and the when of PGS 2.0

STUDY QUESTION: We wanted to probe the opinions and current practices on preimplantation genetic screening (PGS), and more specifically on PGS in its newest form: PGS 2.0? STUDY FINDING: Consensus is lacking on which patient groups, if any at all, can benefit from PGS 2.0 and, a fortiori, whether all IVF patients should be offered PGS. WHAT IS KNOWN ALREADY: It is clear from all experts that PGS 2.0 can be defined as biopsy at the blastocyst stage followed by comprehensive chromosome screening and possibly combined with vitrification. Most agree that mosaicism is less of an issue at the blastocyst stage than at the cleavage stage but whether mosaicism is no issue at all at the blastocyst st…

The Bisphenol A Experience: A Primer for the Analysis of Environmental Effects on Mammalian Reproduction1

It is increasingly evident that environmental factors are a veritable Pandora's box from which new concerns and complications continue to emerge. Although previously considered the domain of toxicologists, it is now clear that an understanding of the effects of the environment on reproduction requires a far broader range of expertise and that, at least for endocrine-disrupting chemicals, many of the tenets of classical toxicology need to be revisited. Indeed, because of the wide range of reproductive effects induced by these chemicals, interest among reproductive biologists has grown rapidly: in 2000, the program for the annual Society for the Study of Reproduction meeting included a single…

High incidence of chromosomal abnormalities in large-headed and multiple-tailed spermatozoa

Rodrigo Vivo, Lorena, lorovi@alumni.uv.es ; Prados Dodd, Nicolas, Nicolas.Prados@uv.es ; Gil Salom, Manuel Luis, Manuel.Gil-Salom@uv.es ; Remohi Gimenez, Jose Alejandro, J.Alejandro.Remohi@uv.es

Intracytoplasmic Testicular Sperm Injection: An Effective Treatment for Otherwise Intractable Obstructive Azoospermia

AbstractPurpose: We evaluated the efficacy of intracytoplasmic sperm injection with testicular spermatozoa.Materials and Methods: Intracytoplasmic sperm injection was performed with spermatozoa obtained from testicular biopsy specimens in 15 patients with obstructive azoospermia, in whom standard microsurgical procedures were not feasible or had previously failed.Results: Fertilization was achieved in 14 to 15 cycles. Mean fertilization rate per cycle was 63.6 percent. Four clinical pregnancies occurred, for a pregnancy rate of 26.7 percent per started cycle and 28.6 percent per transfer.Conclusions: Intracytoplasmic testicular sperm injection is followed by high fertilization rates, and of…

Genetic Analysis of Human Preimplantation Embryos

Preimplantation development comprises the initial stages of mammalian development, before the embryo implants into the mother's uterus. In normal conditions, after fertilization the embryo grows until reaching blastocyst stage. The blastocyst grows as the cells divide and the cavity expands, until it arrives at the uterus, where it "hatches" from the zona pellucida to implant into the uterine wall. Nevertheless, embryo quality and viability can be affected by chromosomal abnormalities, most of which occur during gametogenesis and early embryo development; human embryos produced in vitro are especially vulnerable. Therefore, the selection of chromosomally normal embryos for transfer in assis…

Sperm chromosomal abnormalities and their contribution to human embryo aneuploidy.

In this work we reviewed 18 years of experience using fluorescence in situ hybridization (FISH) for sperm aneuploidy testing. We evaluated parameters associated with increased numerical sperm chromosome abnormalities and determined the male contribution to embryo aneploidies in terms of reproductive outcome by increased sperm aneuploidy. This retrospective study analyzed data from 2008 sperm samples of infertile males undergoing FISH analysis because of clinical history of repetitive implantation failure, recurrent miscarriage, impaired sperm parameters, or mixed causes. Sperm concentration was the only sperm parameter associated with FISH results—we observed a gradual increase of abnormal …

Effect of genistein and daidzein levels on ovarian response and embryo quality in PGT-A cycles

Moderate ovarian stimulation does not increase the incidence of human embryo chromosomal abnormalities in in vitro fertilization cycles.

A high chromosomal abnormalities rate has been observed in human embryos derived from in vitro fertilization (IVF) treatments. The real incidence in natural cycles has been poorly studied, so whether this frequency may be induced by external factors, such as use of gonadotropins for ovarian stimulation, remains unknown.We conducted a prospective cohort study in a University-affiliated private infertility clinic with a comparison between unstimulated and stimulated ovarian cycles in the same women. Preimplantation genetic screening by fluorescence in situ hybridization was performed in all viable d 3 embryos.The primary objective was to compare the incidence of embryo chromosomal abnormaliti…

Preimplantation genetic testing for aneuploidy in patients with partial X monosomy using their own oocytes: is this a suitable indication?

Objective To describe the outcome of preimplantation genetic testing (PGT-A) using their own oocytes in patients with mosaic Turner Syndrome (MTS). The impact of the assisted reproduction technique (ART) performed (PGT-A or oocyte donation) and the type of absence of the X chromosome (total or partial) were considered. Design Retrospective observational multicenter study. Setting University-affiliated private in vitro fertilization center. Patient(s) Fifty-six patients with MTS with whom 65 ovarian stimulation cycles for PGT-A (fluorescence in situ hybridization/arrays-next generation sequencing) were performed. The study included 90 women with MTS and 20 women with pure Turner Syndrome (PT…

Testicular sperm from patients with obstructive and nonobstructive azoospermia: aneuploidy risk and reproductive prognosis using testicular sperm from fertile donors as control samples.

Objective To establish a baseline incidence of chromosomal abnormalities in testicular sperm of fertile men and to determine the best control sample for comparisons with azoospermic males to estimate their reproductive prognosis. Design Prospective study. Setting Infertility clinic. Patient(s) Sixteen obstructive azoospermic (OA) and 19 nonobstructive azoospermic patients (NOA). Control samples were ejaculated sperm from ten fertile donors and testicular sperm from ten other fertile donors. Intervention(s) Fluorescence in situ hybridization (FISH) in sperm. Main Outcome Measure(s) Sperm numerical abnormalities for chromosomes 13, 18, 21, X, and Y; ongoing implantation and pregnancy rates in…

Pregnancy in an azoospermic patient with markedly elevated serum follicle-stimulating hormone levels

Objective To assess the possibility of achieving a pregnancy in an azoospermic patient with markedly elevated serum FSH level. Design A case report. Setting In vitro fertilization program at the Instituto Valenciano de Infertilidad. Patient An azoospermic patient with small testes and serum FSH level (38.7 mlU/mL) higher than three times normal. Testicular biopsy revealed Sertoli cell-only syndrome with focal spermatogenesis. Interventions Intracytoplasmic microinjection of testicular tissue-extracted spermatozoa. Main Outcome Measurements: Fertilization rate, cleavage rate, clinical pregnancy. Results Eight of 11 (73%) intact oocytes showed two pronuclei. All of them cleaved normally. Four…

Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients

Implantation failure (IF) is defined as three or more failed IVF attempts, and preimplantation genetic diagnosis (PGD) is being used in these patients to improve IVF outcome. PGD was performed in 49 implantation failure patients with a mean number of 4.2 +/- 1.6 previous IVF failures, and in nine fertile controls. Fluorescence in-situ hybridization (FISH) on blastomeres from biopsied day 3 embryos was performed for chromosomes 13, 16, 18, 21, 22, X and Y. There was a significantly higher rate of chromosomal abnormalities (67.4%) compared with controls (36.3%). In 57 cycles, a pregnancy rate of 34.0% and an implantation rate of 19.8% was observed in implantation failure patients compared wit…

Prediction model for aneuploidy in early human embryo development revealed by single-cell analysis.

Aneuploidies are prevalent in the human embryo and impair proper development, leading to cell cycle arrest. Recent advances in imaging and molecular and genetic analyses are postulated as promising strategies to unveil the mechanisms involved in aneuploidy generation. Here we combine time-lapse, complete chromosomal assessment and single-cell RT–qPCR to simultaneously obtain information from all cells that compose a human embryo until the approximately eight-cell stage (n=85). Our data indicate that the chromosomal status of aneuploid embryos (n=26), including those that are mosaic (n=3), correlates with significant differences in the duration of the first mitotic phase when compared with e…

Multicenter prospective study of concordance between embryonic cell-free DNA and trophectoderm biopsies from 1301 human blastocysts

Background The recent identification of embryonic cell-free DNA in spent blastocyst media has opened a new era of possibilities for noninvasive embryo aneuploidy testing in assisted reproductive technologies. Yet, previous studies assessing a limited number of embryos reported variable concordance between embryonic cell-free DNA and trophectoderm biopsies, thus questioning the validity of this approach. Objective This study aimed to evaluate the concordance and reproducibility of testing embryonic cell-free DNA vs trophectoderm DNA obtained from the same embryo in a large sample of human blastocysts and to assess the contribution of the inner cell mass and trophectoderm to embryonic cell-fr…

Intracytoplasmic sperm injection: a treatment for extreme oligospermia.

AbstractPurpose: We evaluated the efficacy of intracytoplasmic sperm injection in patients with extreme oligospermia.Materials and Methods: A total of 67 intracytoplasmic sperm injection cycles was attempted in 58 infertile couples in which the husbands had extreme oligospermia (less than 100,000 spermatozoa per ml. ejaculate).Results: Fertilization was achieved in 65 of 67 cycles. Mean fertilization rate per cycle was 66.4 percent. A total of 18 clinical pregnancies was obtained, for a pregnancy rate of 26.8 percent per started cycle. There were 4 miscarriages and 8 live births from 5 deliveries. Nine pregnancies are ongoing.Conclusions: Intracytoplasmic sperm injection in patients with ex…