Albert Obradors

Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: Case Report

Preimplantation genetic diagnosis (PGD) for monogenic diseases is widely applied, allowing the transfer to the uterus of healthy embryos. PGD is also employed for the detection of chromosome abnormalities for couples at high risk of producing aneuploid embryos, such as advanced maternal (>35 years). A significant number of patients requesting PGD for monogenic diseases are also indicated for chromosome testing. We optimized and clinically applied a PGD protocol permitting both cytogenetic and molecular genetic analysis. A couple, carriers of two cystic fibrosis (CF) mutations (c.3849 + 10 KbC > T and c.3408C > A) with a maternal age of 38 years and two previously failed IVF-PGD cycles, was enrolled in the study. After ovarian stimulation, six oocytes were obtained. To detect abnormalities for all 23 chromosomes of the oocyte, the first polar body (1PB) was biopsied from five of the oocytes and analyzed using comparative genomic hybridization (CGH). CGH analysis showed that 1PB 1 and 1PB 4 were aneuploid (22X,-9,-13,+19 and 22X,-6, respectively), while 1PB 2, 1PB 3 and 1PB 6 were euploid. Blastomere biopsy was only applicable on embryos formed from Oocyte 3 and Oocyte 6. After whole-genome amplification with multiple displacement amplification, a multiplex PCR, amplifying informative short tandem repeats (D7S1799; D7S1817) and DNA fragments encompassing the mutation sites, was performed. MiniSequencing was applied to directly detect each mutation. Genetic diagnosis showed that Embryo 6 was affected by CF and Embryo 3 carried only the c.3849 + 10 KbC > T mutation. Embryo 3 was transferred achieving pregnancy and a healthy boy was born. This strategy may lead to increased pregnancy rates by allowing preferential transfer of euploid embryos.

Transvaginal versus transabdominal ultrasound guidance for embryo transfer in donor oocyte recipients: a randomized clinical trial

OBJECTIVE To compare pregnancy and implantation rates with transvaginal (TV) versus transabdominal (TA) ultrasound-guided embryo transfer (ET). DESIGN Randomized, clinical trial registered at clinicaltrials.gov (NCT 01137461). SETTING Private, infertility clinic. PATIENT(S) Three-hundred thirty randomized recipients of donor oocytes. INTERVENTION(S) Embryo transfer using TV (with empty bladder, using the Kitazato ET Long catheter) versus TA ultrasound guidance (with full bladder, using the echogenic Sure View Wallace catheter). MAIN OUTCOME MEASURE(S) Overall pregnancy, clinical pregnancy, implantation, and ongoing pregnancy rates. Duration and difficulty of ET. Patient-reported uterine cramping and discomfort, as evaluated by questionnaire. RESULT(S) No statistically significant differences were observed in clinical pregnancy 50.9% versus 49.4% (95% confidence interval of the difference: -9.2 to +12.2%), implantation 34.5% versus 31.4% (95% CI of the difference: -4 to +10.3%) between the TV and TA ultrasound-guided groups. Transfer difficulty (6% versus 4.2%) and uterine cramping (27.2% versus 18.3%) were not statistically significantly different between treatment groups. Total duration (154±119 versus 85±76 seconds) was statistically significantly higher in the TV ultrasound group. Light to moderate-severe discomfort related to bladder distension was reported by 63% of the patients in the TA ultrasound group. CONCLUSION(S) Transvaginal ultrasound-guided ET yielded similar success rates compared with the TA ultrasound-guided procedure without requiring the assistance of a sonographer. It was associated with increased patient comfort due to the absence of bladder distension.

Comprehensive embryo analysis of advanced maternal age–related aneuploidies and mosaicism by short comparative genomic hybridization

The short comparative genomic hybridization (short-CGH) method was used to perform a comprehensive cytogenetic study of isolated blastomeres from advanced maternal age embryos, discarded after fluorescent in situ hybridization (FISH) preimplantation genetic screening (PGS), detecting aneuploidies (38.5% of which corresponded to chromosomes not screened by 9-chromosome FISH), structural aberrations (31.8%), and mosaicism (77.3%). The short-CGH method was subsequently applied in one PGS, achieving a twin pregnancy.

Reliability of short comparative genomic hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application

BACKGROUND Comparative genomic hybridization (CGH) is a valuable alternative to fluorescence in situ hybridization (FISH) for preimplantation genetic screening (PGS) because it allows full karyotype analysis. However, this approach requires the cryopreservation of biopsied embryos until results are available. The aim of this study is to reduce the hybridization period of CGH, in order to make this short-CGH technique suitable for PGS of Day-3 embryos, avoiding the cryopreservation step. METHODS Thirty-two fibroblasts from six aneuploid cell lines (Coriell) and 48 blastomeres from 10 Day-4 embryos, discarded after PGS by FISH with 9 probes (9-chr-FISH), were analysed by short-CGH. A reanalysis by the standard 72 h-CGH and FISH using telomeric probes was performed when no concordant results between short-CGH and FISH diagnosis were observed. The short-CGH was subsequently applied in a clinical case of advanced maternal age. RESULTS In 100% of the fibroblasts analysed, the characteristic aneuploidies of each cell line were detected by short-CGH. The results of the 48 blastomeres screened by short-CGH were supported by both 72 h-CGH results and FISH reanalysis. The chromosomes most frequently involved in aneuploidy were 22 and 16, but aneuploidies for the other chromosomes, excepting 1, 10 and 13, were also detected. Forty-one of the 94 aneuploid events observed (43.6%) corresponded to chromosomes which are not analysed by 9-chr-FISH. CONCLUSIONS We have performed a preliminary validation of the short-CGH technique, including one clinical case, suggesting this approach may be applied to Day-3 aneuploidy analysis, thereby avoiding embryo cryopreservation and perhaps helping to improve implantation rate after PGS.

Detection of unbalanced chromosome segregations in preimplantation genetic diagnosis of translocations by short comparative genomic hibridization.

OBJECTIVE To apply a comprehensive chromosomal screening through short comparative genomic hybridization (CGH) in the preimplantation genetic diagnosis (PGD) of translocations. DESIGN Clinical research study. SETTING A PGD laboratory and two IVF clinics. PATIENT(S) Three Robertsonian translocation carriers, two reciprocal translocation carriers, and a double-translocation carrier. INTERVENTION(S) After using the short-CGH approach in the reanalysis of two unbalanced embryos, discarded from a PGD for a reciprocal translocation carrier, the same method was applied in the PGD of day-3 embryos of translocation carriers. MAIN OUTCOME MEASURE(S) Ability of short CGH to detect partial chromosomal abnormalities in unbalanced embryos, translocation segregation proportions, and proportion of embryos carrying chromosomal abnormalities not related to the translocations. RESULT(S) The short-CGH technique detected errors resulting from the meiotic segregation of the chromosomes involved in the translocations and other abnormalities affecting the remaining chromosomes. Alternate segregation was detected most frequently among Robertsonian translocation cases, whereas unbalanced chromosome segregations were found predominantly in reciprocal ones. Aneuploidy and structural chromosome errors were found more frequently in Robertsonian than in reciprocal translocation carriers. Application of short-CGH PGD achieved pregnancy in two cases. CONCLUSION(S) Short CGH is a reliable approach for PGD of translocations, as it is capable of detecting partial chromosome errors caused by unbalanced segregations simultaneously to the screening of all chromosomes, and it may improve the results after PGD for translocation carriers.

Outcome of twin babies free of Von Hippel-Lindau disease after a double-factor preimplantation genetic diagnosis: monogenetic mutation analysis and comprehensive aneuploidy screening

OBJECTIVE To increase the embryo implantation rate, a double-factor preimplantation genetic diagnosis (DF-PGD) was performed, selecting for transfer potentially euploid evolved embryos free of the mutation responsible for Von Hippel-Lindau syndrome (VHL). DESIGN Case report. SETTINGS Medical university center and a private IVF center. PATIENT(S) A patient carrier of the R161Q mutation on the VHL gene. INTERVENTION(S) After first polar body (1PB) biopsy, it was analyzed using comparative genomic hybridization (1PB-CGH). On day +3, mutation detection using minisequencing and short tandem repeat analysis was performed in multiple displacement amplification products of a single blastomere per embryo. MAIN OUTCOME MEASURE(S) Transfering embryos free of the disease and originating from euploid oocytes. RESULT(S) Nine of the twelve oocytes obtained were successfully analyzed using 1PB-CGH. One of them was aneuploid (1PB #1: 29XX,+2,+10,+12,+17,+19), and the rest were euploid. All of the oocytes were fertilized and became evolved embryos. Six of the embryos were VHL unaffected and had good quality. Five (83%) of them were potentially euploid. According to cytogenetic results, two of the evolved and healthy embryos were transfered, achieving the birth of healthy twin babies. CONCLUSION(S) The DF-PGD can be a useful tool to increase implantation of transfered embryos in PGD for monogenic diseases.

Paternal age and assisted reproductive outcomes in ICSI donor oocytes: is there an effect of older fathers?

STUDY QUESTION Does paternal age affect semen quality and reproductive outcomes in oocyte donor cycles with ICSI? SUMMARY ANSWER Paternal age is associated with a decrease in sperm quality, however it does not affect either pregnancy or live birth rates in reproductive treatments when the oocytes come from donors <36 years old and ICSI is used. WHAT IS KNOWN ALREADY The weight of evidence suggest that paternal age is associated with decreasing sperm quality, but uncertainty remains as to whether reproductive outcomes are affected. Although developed to treat severe sperm factor infertility, ICSI is gaining popularity and is often used even in the presence of mild male factor infertility. STUDY DESIGN, SIZE, DURATION A retrospective cohort study spanning the period between February 2007 and June 2010. A total of 4887 oocyte donation cycles were included. PARTICIPANTS/MATERIALS, SETTING, METHODS Fertilization was carried out by ICSI in all cycles included, and the semen sample used was from the male partner in all cases. The association of male age with semen parameters (volume, concentration, percentage of motile spermatozoa) was analyzed by multiple analysis of covariance. The association of male age with reproductive outcomes (biochemical pregnancy, miscarriage, ongoing pregnancy and live birth rate) was modeled by logistic regression, where the following covariates were introduced: donor age, recipient age, semen state (fresh versus frozen) and number of transferred embryos (3 and 2 versus 1). MAIN RESULTS AND THE ROLE OF CHANCE We identified a significant relationship between paternal age and all sperm parameters analyzed: for every 5 years of age, sperm volume decreases by 0.22 ml (P < 0.001), concentration increases by 3.1 million sperm/ml (P = 0.003) and percentage motile spermatozoa decreases by 1.2% (P < 0.001). No differences were found in reproductive outcomes (biochemical pregnancy, miscarriage, clinical pregnancy, ongoing pregnancy and live birth) among different male age groups. LIMITATIONS, REASONS FOR CAUTION The use of donor oocytes, while extremely useful in highlighting the role of male age in reproductive outcomes, limits the generalization of our results to a population of young women with older male partners. No data were available on perinatal and obstetrical outcomes of these pregnancies. Most (75%) cycles used frozen/thawed sperm samples which might have introduced a bias owing to loss of viability after thawing. ICSI was performed in all cycles to control for fertilization method; this technique could mask the natural fertilization rate of poorer sperm samples. Furthermore, we did not use stringent ICSI indications; and our data are therefore not generalizable to cases where only severe male factor is considered. However, male patients were of different racial background, thus allowing generalizing our results to a wider patient base. WIDER IMPLICATIONS OF THE FINDINGS Our study suggests that paternal age does not affect reproductive outcomes when the oocyte donor is <36 years of age, indicating that ICSI and oocyte quality can jointly overcome the lower reproductive potential of older semen. STUDY FUNDING/COMPETING INTEREST(S) This study was supported in part by Fundació Privada EUGIN. The authors have no conflicts of interest to declare.

Errors at mitotic segregation early in oogenesis and at first meiotic division in oocytes from donor females: Comparative genomic hybridization analyses in metaphase II oocytes and their first polar body

The aim of this work is to analyze, using the comparative genomic hybridization technique, the frequencies and the mechanisms involved in the production of aneuploidy events in donor oocytes. The results showed that 32.1% of them were aneuploid, with 51.7% of those originating from first meiotic division errors and 48.3% from the presence of aneuploid oogonium.

First successful double‐factor PGD for Lynch syndrome: monogenic analysis and comprehensive aneuploidy screening

Preimplantation genetic diagnosis (PGD) has been applied worldwide for a great variety of single‐gene disorders over the last 20 years. The aim of this work was to perform a double‐factor preimplantation genetic diagnosis (DF‐PGD) protocol in a family at risk for Lynch syndrome. The family underwent a DF‐PGD approach in which two blastomeres from each cleavage‐stage embryo were biopsied and used for monogenic and comprehensive cytogenetic analysis, respectively. Fourteen embryos were biopsied for the monogenic disease and after multiple displacement amplification (MDA), 12 embryos were diagnosed; 5 being non‐affected and 7 affected by the disease. Thirteen were biopsied to perform the aneuploidy screening by short‐comparative genomic hybridization (CGH). The improved DF‐PGD approach permitted the selection of not only healthy but also euploid embryos for transfer. This has been the first time a double analysis of embryos has been performed in a family affected by Lynch syndrome, resulting in the birth of two healthy children. The protocol described in this work offers a reliable alternative for single‐gene disorder assessment together with a comprehensive aneuploidy screening of the embryos that may increase the chances of pregnancy and birth of transferred embryos.

Non-meiotic chromosome instability in human immature oocytes.

Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25–45 years of age) and 24 IVF oocyte donors (18–33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes.