Embryo aneuploidy screening for repeated implantation failure and unexplained recurrent miscarriage.

Among other factors, chromosomal abnormalities that originate from gametogenesis and preimplantation embryonic development are thought to be one of the major contributing factors for early embryonic death and failure of pregnancy. However, so far, no non-invasive technique exists that allows the detection of the chromosomal complement of an oocyte or a developing embryo as a whole. Rather, by removing polar bodies/blastomeres, recent developments on preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) have paved the way to detect and possibly eliminate the majority of chromosomally abnormal embryos, thereby increasing the chance of a healthy pregnancy. This article summarizes the origin and impact of chromosomal abnormalities on human reproduction in cases with repeated implantation failure (RIF) and unexplained recurrent miscarriage. It also discusses recent advances regarding the possible benefits of PGD-AS in such cases.

Preliminary FISH studies on spermatozoa and embryos in patients with variable degrees of teratozoospermia and a history of poor prognosis.

The aim of this study was to analyse to what extent sperm aneuploidy is associated with sperm morphology and subsequently with embryo aneuploidy. Fifty-nine men with variable degrees of teratozoospermia and previously poor assisted reproduction prognosis were included in the study. Samples from 10 normozoospermic men with proven fertility were used as controls. Individual spermatozoa were scored for chromosomes 13, 21 and for 18, X, Y separately. Compared with controls, 23 out of 59 cases (39.0%) were found to have increased sperm aneuploidy for at least one of the chromosomes analysed in a treatment cycle. Fifty-two patients underwent a treatment cycle and were documented according to the pregnancy and spermatozoa fluorescence in-situ hybridization results. A total of 121 previous unsuccessful assisted reproduction cycles of the cases were then retrospectively reviewed. In 23 of the latest cycles, preimplantation genetic diagnosis was applied to 106 cleavage stage embryos and 47 of 94 embryos analysed (50.0%) were found to be chromosomally abnormal. Furthermore, 16 of 47 (34.0%) embryos with chromosomal abnormality were carrying complex chromosomal defects. The results imply that increased aneuploidy is present in both spermatozoa and embryos in couples with severe male infertility with a history of repeated unsuccessful attempts. Therefore, proper genetic counselling should be considered in these cases.

Clinical aspects of preimplantation genetic diagnosis for single gene disorders combined with HLA typing.

Preimplantation genetic diagnosis (PGD) for single gene disorders combined with human leukocyte antigen (HLA) matching has recently emerged as a therapeutic tool for stem cell transplantation in couples bearing an affected offspring. There may exist, however, several patient- or cycle-specific limitations for certain couples. This article documents data regarding experience of single gene disorders combined with HLA matching obtained at Istanbul Memorial Hospital, Turkey. The data were obtained from 20 couples undergoing 26 PGD-HLA cycles for thalassaemia (n = 23), Wiscott-Aldrich syndrome (n = 1) and acute lymphoblastic leukaemia (n = 2). A total of 206 embryos was biopsied on day 3 of embryo development and subsequently analysed. After the analysis, 26 (12.6%) of them were found to be both healthy and HLA compatible. In 16 embryo transfers performed, seven (43.7%) clinical pregnancies were obtained, one of which resulted in miscarriage. Ten of the 26 cycles started (38.4%) were cancelled due to a lack of suitable (mutation-free and/or HLA-compatible) embryos. The data suggest that application of PGD in combination with HLA typing is a promising therapeutic tool for an affected sibling.

Effect of PGD on implantation and ongoing pregnancy rates in cases with predominantly macrocephalic spermatozoa.

Although its occurrence is rare, the presence of large headed or macrocephalic spermatozoa and increased chromosomal abnormality has recently been reported by several groups. Moreover, when intracytoplasmic sperm injection (ICSI) was performed with samples containing macrocephalic spermatozoa, lower fertilization and implantation rates result in poor clinical outcome. In order to evaluate the impact of preimplantation genetic diagnosis (PGD) on implantation and ongoing pregnancy rates in these couples, the results of 23 PGD cycles were compared with non-PGD cycles (n = 60) as well as cycles with absolute teratozoospermia (having zero normal morphology) with (n = 14) or without PGD (n = 66). Out of 82 embryos biopsied in the macrocephalic sperm group, abnormalities were detected in 46.4% of the embryos analysed. Most of the abnormalities were trisomies (37.0%) and complex aneuploidies (51.9%). A 33.3% pregnancy rate was achieved by selectively transferring euploid embryos after PGD with the statistically higher implantation rate of 25.0% compared with non-PGD cycles (IR: 12.3%, P < 0.01). Moreover, only one missed abortion (14.3%) was observed in the PGD group, whereas seven of the 15 pregnancies resulted in abortion in the non-PGD group (46.7%). Preliminary results indicate that patients should be counselled for increased chromosomal abnormality and a possible beneficial effect of eliminating chromosomally abnormal embryos with PGD on a bortion rates.

The results of aneuploidy screening in 276 couples undergoing assisted reproductive techniques.

Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) using sequential in situ hybridization was applied for aneuploidy testing in 276 couples with 282 ART cycles. Patients with advanced maternal age (AMA, n = 147), recurrent implantation failure (RIF, n = 48), repeated early spontaneous abortion (RSA, n = 32) and abnormal gamete cell morphology (AGCM, n = 55) including macrocephal sperm forms or cytoplasmic granular oocytes were included. Embryo biopsy was performed on day 3 in a calcium-magnesium-free medium by using a noncontact diode laser system. After fixation and enzymatic treatment, fluorescent in situ hybridization (FISH) was carried out on 1147 blastomeres with specific probes for chromosomes 13, 16, 18, 21 and 22 for AMA group, 13, 18, 21, X and Y for AGCM group and 13, 16, 18, 21, 22, X and Y for RIF and RSA groups respectively. The overall chromosomal abnormality rate in analyzed embryos was 40.9%, with no significant difference between AMA, RIF and RSA groups (p > 0.05). However, AGCM group presented a higher rate of chromosomal aneuploidies (57.4%) than the other three groups (p < 0.01). A total of 84% biopsied embryos presented cleavage in 24 h and embryo transfer was realized in 278 cycles. In four cycles, no chromosomally normal embryo was found for embryo transfer. A total of 88 pregnancies (31.6%) were achieved, 19.3% resulted in abortion and 63 healthy births were obtained, with a total of 93 babies born. Aneuploidy testing in couples with poor prognosis undergoing ART cycles is a useful tool to increase the chance of ART success. Furthermore, abnormal gamete cell morphology should be considered one of the major indications for PGD in ART programs as high aneuploidy rates were observed in this group.

Development and clinical application of a strategy for preimplantation genetic diagnosis of single gene disorders combined with HLA matching.

Preimplantation HLA matching has recently emerged as a tool for couples desiring to conceive a potential donor progeny for transplantation in a sibling with a life-threatening disorder. In this paper we describe a strategy optimized for preimplantation genetic diagnosis (PGD) of haemoglobinopathies combined with HLA matching. This procedure involves a minisequencing-based genotyping of HLA regions A, B, C and DRB combined with mutation analysis of the gene regions involved by mutation. Analysis of at least eight polymorphic short tandem repeat (STR) markers scattered through the HLA complex has also been included to detect potential contamination and crossing-over occurrences between HLA genes. The above assay can also be used for preimplantation HLA matching as a primary indication. The strategy was clinically applied for HLA matching in 17 cycles (14 for beta-thalassaemia, one for Wiscott-Aldrich syndrome and two for leukaemia). A reliable HLA genotype was achieved in 255/266 (95.9%) of the blastomeres. In total, 22 (14.8%) embryos were obtained that were HLA-matched with the affected siblings, 14 (9.4%) of which were unaffected and transferred back to the patients. Four clinical pregnancies were obtained, three of which (one twin, two singletons) are ongoing and were confirmed as healthy and HLA-identical with the affected children. Minisequencing-based HLA typing combined with HLA STR haplotyping has been shown to be a reliable strategy for preimplantation HLA matching. The major advantage of this approach is that the validation of a single assay can be done once and then used for the majority of the patients, reducing notably time needed for preclinical set-up of each case.

Assessment of DNA fragmentation and aneuploidy on poor quality human embryos.

In human assisted reproduction, low embryo quality due to retarded growth and abnormal cellular morphology results in fewer embryos suitable for transfer. This study aimed to assess the extent of DNA fragmentation and aneuploidy in spare slow growing or arrested human embryos. In 19 assisted reproduction cycles, a total of 57 embryos unsuitable for embryo transfer were used for simultaneous apoptosis and aneuploidy assessment. Among them, 31 (54.3%) showed DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) analysis. Among 26 embryos that were negative for TUNEL, interpretable fluorescence in-situ hybridization (FISH) results were obtained for 25 embryos (96.2%). Sixteen embryos were detected to be chromosomally abnormal (64.0%); three were found to be chaotic, six had complex aneuploidy, six had complete monosomy and one was polyploid. The results show that a high level of DNA fragmentation and aneuploidy are common in embryos with slow growth and/or low quality. More detailed studies are needed to assess the effect of factors such as ovarian stimulation regimens and in-vitro culture conditions. Moreover, application of simultaneous TUNEL and FISH techniques can be informative regarding DNA integrity and aneuploidy.

Embryo development characteristics in Robertsonian and reciprocal translocations: a comparison of results with non-translocation cases.

The effect of translocations on embryo development was evaluated and results were compared in terms of embryo development with those of embryos obtained from standard intracytoplasmic sperm injection (ICSI) cycles. In 23 translocation carriers with 34 cycles, fertilization, pronuclear morphology scoring (PMS), developmental arrest, cleavage and blastocyst formation were evaluated and compared with embryos obtained from non-translocation cases undergoing ICSI (n = 98 cycles). In 28 cycles, preimplantation genetic diagnosis (PGD) was performed on prezygotes (first and second polar body biopsy for female carriers; n = 3) or on embryos having seven or more blastomeres (blastomere biopsy; n = 25). In six cycles for four couples, probes for translocated chromosomes were not available, so PGD could not be performed. Overall, in translocation cases, a lower fertilization rate, a higher rate of retarded embryo development, and a lower rate of blastocyst formation were observed compared with embryos of non-translocation cases. Fluorescence in-situ hybridization (FISH) analysis showed a 70.9% abnormality rate for reciprocal translocations and 55.0% for Robertsonian translocations respectively. In cases with Robertsonian and reciprocal translocation carriers, the probability of poor embryo development, which may be a result of high segregation abnormalities, may negatively affect the outcome of assisted reproductive techniques. This poor prognosis should also be considered when genetic counselling for translocation is given.

Results of preimplantation genetic diagnosis in patients with Klinefelter's syndrome.

With the application of preimplantation genetic diagnosis (PGD), a possible genetic contribution of spermatozoa obtained from 47,XXY non-mosaic Klinefelter patients on preimplantation embryos was analysed in eight couples. Interpretable fluorescence in-situ hybridization results were obtained for 28 out of 33 embryos biopsied (84.8%) and 23 blastomeres were analysed for chromosomes 13, 18, 21, X and Y. Nine out of 23 embryos were diagnosed as abnormal (39.1%). Five out of nine contained sex chromosome abnormalities (55.5%). Two were diagnosed as 47,XXY and three were found to have monosomy X. Besides sex chromosomal abnormalities, other abnormalities detected were haploidy, triploidy, monosomy 13, monosomy 18 and trisomy 13. Five blastomeres were analysed for sex chromosomes only and all of them were found to be normal. Overall, the rate of sex chromosome abnormality in biopsied embryos was found to be 17.8% (5/28). Moreover, among 33 embryos biopsied, five of the eight zygotes, which were classified as a poor prognosis group according to pronuclear morphology scoring, showed an impaired growth profile after biopsy and were found to be chromosomally abnormal. Elimination of abnormal embyos and transfer of normal ones resulted in four pregnancies in eight cycles (50%). Two pregnancies, one singleton and one twins resulted in healthy births. Two pregnancies, one singleton and one twins are continuing beyond the second trimester. These results show that there is in fact elevated chromosomal abnormality for both sex chromosomes and autosomes in embryos developed from Klinefelter males. Furthermore together with PGD, embryo scoring according to pronuclear morphology can give additional benefit for selecting chromosomally abnormal embryos. Therefore, PGD should be recommended in cases with Klinefelter's syndrome and this information should be discussed with the couple when genetic counselling is given.

Pronuclear morphology scoring and chromosomal status of embryos in severe male infertility.

BACKGROUND: The study aim was to evaluate the relationship between pronuclei morphology scoring (PNMS) and the chromosomal complement of embryos in couples with severe male infertility undergoing ICSI. A total of 3116 pre-embryos was scored according to PNMS in 452 cycles. METHODS: Pre-embryos were classified into eight categories based on the alignment, size, linear or irregular distribution of pronuclear bodies (PNB), position and clarity of cytoplasmic halo and abutting of the pronucleus. These categories were subdivided into groups I and II according to the similarity and distribution of PNB. RESULTS: In total, 2574 pre-embryos formed by using ejaculated sperm, while 542 pre-embryos developed by injection of testicular sperm or round spermatids. More group II pre-embryos with markedly different morphology from group I were formed after ICSI with testicular sperm than with fresh ejaculated sperm (32.1 versus 22.7%, P < 0.01). Of 490 pre-embryos in which pronuclear morphology was evaluated, 263 were biopsied for preimplantation genetic diagnosis. The rate of chromosomal abnormality was higher in embryos developed from group II pre-embryos (52.2%) than in embryos developed from group I prezygotes (37.6%, P < 0.05). CONCLUSIONS: Group II pre-embryos had markedly different morphology from group I, and had a low rate of blastocyst formation and high risk of chromosomally abnormal embryos. When testicular sperm and round spermatids were used for ICSI, more group II pre-embryos and chromosomally abnormal embryos were produced than with ejaculated sperm. Pronuclear morphology was correlated with chromosomal complement, and impacted upon by the sperm source.

A comparative study of three techniques for the analysis of sperm recovery: touch-print cytology, wet preparation, and testicular histopathology.

PURPOSE: The aim of this study is to evaluate the efficacy of simultaneous testicular touch-print cytology, testicular histopathology, and wet preparation in nonobstructive azoospermic (NOA) males. METHODS: Three hundred and sixty-three males with NOA underwent a multiple testicular sampling prior to ICSI for histopathologic evaluation, diagnostic testicular sperm extraction, and simultaneous touch-print cytology to evaluate sperm presence or absence. A total of 979 testicular samples were taken. RESULTS: Sperm recovery was achieved in 106 cases (29.2%). Patients with hypospermatogenesis and focal spermatogenesis needed 2.8 and 5.9 biopsies, respectively, to retrieve spermatozoa, while in patients with germ cell aplasia and maturation arrest, even after eight to nine samples no spermatozoa were recovered. Neither the FSH levels nor the testicular volume was found to be significant in the prediction of sperm recovery. If only a single testis was to be biopsied, 25% of the cases with sperm recovery would have been missed. The combination of touch-print cytology with histopathology and wet preparation increased the accuracy of spermatozoa identification. CONCLUSION: Touch-print cytology was found to be more predictive than wet preparation in the diagnosis of spermatogenesis; moreover, it was found to be a quick and easy technique providing an accurate diagnosis in prediction of sperm recovery.

Relationship between granular cytoplasm of oocytes and pregnancy outcome following intracytoplasmic sperm injection.

Couples undergoing intracytoplasmic sperm injection (ICSI) for male infertility using oocytes with centrally located granular cytoplasm (CLCG) were evaluated for fertilization, embryo development, implantation and pregnancy rate. CLCG is a rare morphological feature of the oocyte, that is diagnosed as a larger, dark, spongy granular area in the cytoplasm. Severity is based on both the diameter of granular area and the depth of the lesion. Twenty-seven couples with 39 cycles presenting CLCG in >50% of retrieved oocytes were evaluated. A total of 489 oocytes was retrieved, out of which 392 were at MII. CLCG was observed in 258 of the MII oocytes (65. 8%); 66.7% of these oocytes had slight and 33.3% had severe CLCG. The overall fertilization rate was 72.2% and no statistical significant difference was found between normal and CLCG oocytes and between the oocytes representing slight and severe CLCG. The development and quality of embryos was the same in normal and CLCG oocytes. In nine cycles, preimplantation genetic diagnosis was executed to evaluate a possible accompanying chromosomal abnormality. Out of 44 blastomeres biopsied, 23 had chromosomal abnormality (52. 3%). Eleven pregnancies were achieved in 39 cycles (28.2%), six pregnancies resulted in abortion (54.5%). The implantation rate was found to be 4.2%. Only five ongoing pregnancies were achieved in 39 cycles (12.8%). Couples with CLCG oocytes should be informed about poor on-going pregnancy rates even if fertilization, embryo quality and total pregnancy rates are normal. Furthermore, a high aneuploidy rate may be linked to a high abortion rate.