The hexanucleotide expansion at C9orf72 is the most common genetic alteration in Familial Amyotrophic Lateral Sclerosis (ALS)/Frontotemporal Dementia (FTD). Objectives were to design C9orf72-Exp PGD, perform IVF with C9orf72-Exp PGD to obtain offspring from unaffected embryos, derive C9orf72-Exp ds-hESCs and place ds-hESC line(s) on the National Institutes of Health (NIH) hESC registry making them available for Federally-funded research. Public-private collaboration for PGD assay design, application to IVF and C9orf72-Exp ds-hESC derivation. A couple without infertility, with a family history of FTD caused by C9orf72-Exp, proactively requested IVF with PGD/aneuploidy screening, elective single embryo transfer (eSET) with unaffected euploid embryos and donation of C9orf72-Exp embryos for ds-hESC derivation. PGD was designed with ALS/FTD at-risk couple informative short tandem repeat (STR) markers linked to the pathogenic C9orf72 allele. Blood samples from the couple and an affected grandparent were used to design primers for identification of the expansion haplotype in embryos. Following IVF, blastocyst biopsies were used for whole genome amplification, PGD, 24-chromosome microarray and blastocysts vitrified. A warmed euploid unaffected blastocyst was used for eSET. Inner cell masses of affected blastocysts were cultured on human foreskin fibroblast in xeno-free DMEM + fibroblast growth factor for ds-hESC derivation/growth. Presumptive ds-hESCs were grown for 20 passages, karyotyped, assessed for pluripotency, tested for C9orf72-Exp and embryoid bodies assessed for markers of endoderm, mesoderm, and ectoderm. Embryo donation and ds-hESC derivation documents were submitted to NIH and reviewed for compliance. Eight biopsies had C9orf72-Exp PGD and aneuploidy screening resulting in six C9orf72-Exp affected and two unaffected euploid embryos. One unaffected euploid blastocyst used in eSET resulted in birth of a healthy child. Of the six C9orf72-Exp affected embryos, four were chromosomally normal and available for ds-hESC derivation. One C9orf72-Exp affected blastocyst was successfully used to establish a ds-hESC line with normal karyotype and retention of the C9orf72-Exp. Documents demonstrated compliance with all 15 elements of the NIH Guidelines for Human Stem Cell Research, and the C9orf72-Exp ds-hESC was accepted on the NIH hESC registry. This report details a collaborative pathway providing couples an opportunity to: i) build a family without continuation of a monogenic familial disease; ii) donate otherwise discarded embryos for derivation of ds-hESCs; and thus iii) establish a universally-shared resource to study molecular causes and consequences of familial monogenic diseases and discover future treatments and/or cures of diseases that affect(ed) their living/deceased relatives.

Vitrification using the closed CryoTip system was compared with conventional slow cooling for the purposes of cryopreservation of supernumerary blastocysts after In Vitro Fertilization cycles. Results were compared from embryo thaw cycles between the two groups during a concurrent time period. Vitrification was found to be more effective than slow cooling. Both embryo survival and pregnancy rates were higher when blastocysts were cryopreserved using vitrification.

To determine if there was a correlation between embryo progression at the time of biopsy and the outcome of aneuploidy screening, we investigated the proportion of normal, abnormal and undiagnosed embryos as compared to the cell stage at embryo biopsy. A retrospective study of embryos biopsied for the purpose of aneuploidy screening from May, 2005 through February, 2007. Embryos from patients in our preimplantation genetic diagnosis (PGD) program were biopsied and tested for aneuploidy of 8 chromosomes (13, 15, 16, 18, 21, 22, X and Y). Indications for aneuploidy screening by fluorescence in situ hybridization (FISH) included advanced maternal age, recurrent pregnancy loss, previous pregnancy with aneuploidy, and recurrent IVF failure. A single cell was removed from each embryo that had progressed to at least 5 cells on the morning of day 3 using either mechanical or laser biopsy techniques. Cells were scored as normal (a nucleus with two FISH signals for each autosome and a pair of sex chromosomes), abnormal (a nucleus with extra or missing copies of at least a single chromosome), or undetermined (a nucleus that could not be diagnosed). Regression analysis was performed on the three study groups to determine if there was a correlation between cell stage at biopsy and outcome following PGD. A total of 1011 embryos were biopsied during this time period. The results of the study can be seen in Table 1.Tabled 1Table# Normal (%)# Abnormal (%)# Undiagnosed (%)Total # Embryos5-cell embryos49 (35.3)57 (41.0)33 (23.7)1396-cell embryos72 (45.0)63 (39.4)25 (15.6)1607-cell embryos119 (49.4)93 (38.6)29 (12.0)2418-cell embryos183 (51.4)132 (37.1)41 (11.5)3569+cell embryos52 (45.2)47 (40.9)16 (13.9)115P values0.001<0.001N.S. Open table in a new tab Cell stage at embryo biopsy is positively correlated with diagnostic outcome following PGD. Embryos with better progression on day 3 had a higher likelihood of having a normal FISH result. Embryos with the poorest progression (most notably 5-cell embryos) had a lower likelihood of a normal FISH result and, although not statistically significant, a higher proportion of these embryos went undiagnosed. Slow embryo growth in vitro may be an indication of cytogenetic abnormalities or other intrinsic weakness in these embryos. Further studies on progression following biopsy and resulting pregnancies are underway.

Objective: To evaluate the clinical outcome of in vitro fertilization (IVF) treatment cycles from individual oocyte donors who underwent multiple sequential donations.Methods: We reviewed clinical outcome data from sequential anonymous oocyte donation cycles using donors who underwent multiple IVF stimulations. Donors were grouped by the interval between cycles and the cycle number (rank). The primary outcome measure was delivery rate by individual donor per retrieval from the combined derivative fresh and frozen embryo transfers.Results: Duration and amount of gonadotropin therapy and the fertilization rates did not correlate significantly with the interval between cycles or cycle rank. Cumulative delivered pregnancy rates for cycles 1-6 were 51.5%, 54.6%, 50.5%, 51.5%, 51.1%, and 57.6%, respectively. Delivered pregnancy rates did not vary by interval between cycles.Conclusion: Young healthy presumed or proven fertile women can reliably donate oocytes for at least six cycles with the expectation of consistently high pregnancy rates. (Obstet Gynecol 2001;97:201-4. (C) 2001 by The American College of Obstetricians and Gynecologists.).

Objective: Couples with children who have spinal muscular atrophy type I (SMA) face a 25% risk of having affected offspring with spontaneous conception. Preimplantation genetic testing (PGT) is possible for the deletions in the survival motor neuron (SMN) gene that have been identified in 98% of SMA type I cases. PGT would provide new reproductive options for families at risk for SMA. Methods: Three couples with previously affected children confirmed by DNA testing each underwent in vitro fertilization (IVF) and PGT of the resulting embryos. One or two blastomeres were biopsied from each embryo and analyzed for deletions in exons 7 and 8 of the SMN gene. Results: Nine embryos were predicted to be unaffected, three to be affected, and one embryo could not be interpreted. One of three patients receiving transfer of unaffected embryos became pregnant with twins. Conclusions: Preimplantation genetic testing provides a means for couples at risk for spinal muscular atrophy type I to reduce their chance of initiating an affected pregnancy.

To determine whether ultrasonographic findings can predict the karyotype of spontaneous abortions, 137 pregnancies (54 spontaneous, 83 assisted ovulatory cycles) that subsequently aborted and had chromosome analysis performed on the products of conception were studied ultrasonographically, Transvaginal ultrasound was performed using an Acuson 128XP/10 with 7.5 MHz probe, The numbers of empty gestational sacs, small and normal for gestational size, embryonic poles and embryos with documented cardiac activity were calculated, The frequency of each of these findings in pregnancies with normal and abnormal karyotypes was compared, Of the 137 spontaneous abortions, 51 had normal chromosome analyses and 86 had abnormal karyotypes (68 aneuploidies and 18 polyploidies), Ultrasonographic findings in the 51 karyotypically normal pregnancies included 16 (31%) with empty gestational sacs, and 35 (69%) with embryonic poles, of which 24 (69%) were at least 1 week smaller than expected for gestational age and 11 (31%) were the expected size, Embryonic cardiac activity was documented in 22 (63%) of the 35 embryonic poles, Amongst 86 pregnancies with abnormal karyotypes, similar frequencies of ultrasound findings were found: 23 (27%) with empty gestational sacs, 42 (67%) with embryonic poles smaller than expected for gestational age, and 50 (79%) embryos lost after documentation of embryonic cardiac activity, No differences in the frequency of ultrasonographic findings of empty gestational sacs, small embryonic pole and embryonic cardiac activity were observed between karyotypically normal and abnormal spontaneous abortions, Ultrasonographic findings cannot predict the karyotype of spontaneous abortions.

Objective: To compare outcome of pregnancies after intracytoplasmic sperm injection (ICSI) with those of other assisted reproductive technologies.Design: Pregnancy outcomes after ICSI were followed prospectively and compared with pregnancy outcomes after IVF with fresh and frozen ETs and donor oocyte cycles.Setting: A private tertiary referral center for genetics and infertility in Fairfax, Virginia.Patients: One hundred thirty-six couples achieving pregnancy after undergoing ICSI, 71 after IVF, 35 donor oocyte recipients, and 19 after transfer of frozen-thawed embryos.Interventions: In vitro fertilization and/or ET for all couples. Dilatation and curettage to obtain products of conception for chromosome analysis in 28 women experiencing spontaneous abortion.Main Outcome Measures: Pregnancy outcomes were classified as preclinical loss, clinical loss, and ongoing pregnancy.Results: The mean frequency of preclinical pregnancy loss was 26% after ICSI, 28% after IVF, 3% after ET using donor oocytes, and 11% after frozen ET. The rate of clinical loss after ICSI (21%) was compared with IVF (18%), donor oocyte cycles (11%), and frozen ETs (21%).Conclusions: Intracytoplasmic sperm injection is not associated with an increase in pregnancy losses, clinical or preclinical, compared with conventional IVF.

Objective: To evaluate, in a prospective study, the fertilization and pregnancy rates after intracytoplasmic sperm injection (ICSI) in infertile couples with severe male infertility.Design: Intracytoplasmic sperm injection was performed in 229 consecutive IVF cycles on 190 couples with rigorously defined severe male infertility or proven failure of fertilization in prior NF cycles. Neither male nor female partners were chosen from a waiting list or on any other selective basis, including age, prior or anticipated ovarian response, or oocyte number or quality. There were no upper age limits, in no instance was donor sperm used for ICSI, and cycle cancellation rate was minimal.Setting: Private genetics and fertility center in Fairfax, Virginia.Main Outcome Measures: Fertilization, transfer, and pregnancy rates were measured in ICSI-treated couples, and comparisons were made regarding both female age and strictly defined semen categories.Results: Two hundred six cycles (90%) resulted in ETs, with initiation of 52 pregnancies (25% per transfer, 23% per cycle). Thirty-eight of 52 (18% per transfer) were clinical pregnancies with established gestational sacs or were ongoing or delivered. Pregnancies were achieved even in older women but were more readily established in younger women producing larger numbers of metaphase II oocytes. The severity of semen abnormalities had some small effect on fertilization rate, but only actual necrospermia was associated with markedly decreased frequency of embryo formation. Pregnancy per transfer was similar across groups. In some cases, pregnancy was initiated with fewer than 100 viable sperm in the ejaculate.Conclusions: Intracytoplasmic sperm injection is a very powerful new treatment for severe male infertility. Paradoxically, egg number and probably egg quality are now the main determinants of success in treating male infertility.

Four cases having mosaicism for a small marker or ring [45,X/46,X,+mar or 45,X/46,X,+r] chromosome were ascertained following cytogenetic studies requested because of minor anomalies (cases 1, 3, and 4) and/or short stature (cases 2 and 4). While all 4 cases had traits typical of Ullrich-Turner syndrome (UTS), cases 1, 3, and 4 had manifestations not usually present in UTS, including unusual facial appearance, mental retardation/developmental delay (MR/DD) (cases 3 and 4), and syndactylies (case 1). The facial appearances of cases 1 and 3 were similar yet distinct from that of case 4. Using fluorescence in situ hybridization (FISH), each of the markers in these 4 cases was identified as having been derived from an X chromosome. The level of mosaicism for the mar/r(X) cell line in these cases varied from 70% (case 1) to 16% (case 4) but was not apparently correlated with the presence of MR/DD. Replication studies demonstrated a probable early replication pattern for the mar/r(X) in cases 1, 3, and 4, while the marker in case 2 was apparently late replicating. To date, 41 individuals having mosaicism for a small mar/r(X) chromosome have been described. Interestingly, most of the 14 individuals having a presumedly active mar/r(X) demonstrated clinical findings atypical of UTS, including abnormal facial changes (11) and MR/DD (13). MR was noted most frequently in those cases having at least 50% mosaicism for the marker or ring. In contrast, atypical UTS facial appearance or MR/DD was not noted in 14 of the 16 cases with UTS who carried a probable late replicating marker or ring. In conclusion, although the phenotype of 45,X/46,X,mar/r(X) individuals appears to be influenced by the genetic content and degree of mosaicism for the mar/r(X), the most significant factor associated with MR/DD appears to be the activity status of the mar/r(X) chromosome. Thus, our 4 cases provide further support for the hypothesis that a lack of inactivation of a small mar/r(X) chromosome may be a factor leading to the MR and other phenotypic abnormalities seen in this subset of individuals having atypical UTS.

Human X- and Y-chromosome-bearing spermatozoa were separated based on their DNA content, using modified flow cytometric cell sorting technology. The resulting separation purity of the X-bearing from Y-bearing spermatozoa was evaluated using in-situ hybridization with alpha satellite DNA probes for the X- and Y-chromosomes. In the putative X-enriched-sorted populations, an average of 82% of the spermatozoa showed a hybridization signal with the X probe. Similarly, in the Y-sorted population 75% gave a signal with the Y probe. Sorted X- and Y-bearing spermatozoa were found to maintain their viability for several hours after sorting. These results demonstrate that the human sperm sex ratio can be significantly shifted to favour the selection of female-producing (X) spermatozoa or male-producing (Y) spermatozoa when spermatozoa are flow cytometrically sorted on the basis of DNA content. We propose that flow cytometrically sorted human spermatozoa, used in conjunction with in-vitro fertilization or intra-oviductal insemination, could be used by families who are at risk for X-linked diseases to preferentially produce female offspring. Sorted spermatozoa could also be used to pre-select for male offspring if that were medically indicated.

We prospectively studied the ability of acrosome reaction (AR) inducibility to predict fertilization success in a group of 232 infertile patients presenting sequentially for in-vitro fertilization (IVF). The median percentage of eggs fertilized for the overall patient population was 25% (interquartile range 5-58%), with one to 29 oocytes available for insemination (median, five oocytes). The median percentage of eggs fertilized at IVF increased as the percentage of spermatozoa able to undergo AR became greater: spermatozoa with a failed AR (< or = 5%) fertilized only 12% of eggs, while spermatozoa with AR values > 9% fertilized 50% of eggs. The assay had a specificity of 0.75, a sensitivity of 0.55 and an odds ratio of 2.9; thus, AR-positive patients are 2.9 times more likely to achieve fertilization than patients with a failed AR. Receiver operator characteristic (ROC) curves were constructed for AR, sperm concentration and percentage of normal forms in semen. All three parameters proved to be potentially useful in predicting the occurrence of fertilization, although AR and morphology appeared to be better than sperm concentration by ROC analysis. Patients were divided into four clearly defined subgroups according to their traditional semen characteristics, including morphology. The median percentage of eggs fertilized decreased as traditional semen characteristics deteriorated, from a median of 46% for patients with excellent sperm concentration, motility and morphology, to a median of 29% for patients with suboptimal semen quality and a median of 0% for patients with severely impaired semen.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytogenetic data are presented for 11 473 chorionic villus sampling (CVS) procedures from nine centres in the U.S. NICHD collaborative study. A successful cytogenetic diagnosis was obtained in 99.7 per cent of cases, with data obtained from the direct method only (26 per cent), culture method only (42 per cent), or a combination of both (32 per cent). A total of 1.1 per cent of patients had a second CVS or amniocentesis procedure for reasons related to the cytogenetic diagnostic procedure, including laboratory failures (27 cases), maternal cell contamination (4 cases), or mosaic or ambiguous cytogenetic results (98 cases). There were no diagnostic errors involving trisomies for chromosomes 21, 18, and 13. For sex chromosome aneuploidies, one patient terminated her pregnancy on the basis of non‐mosaic 47,XXX in the direct method prior to the availability of results from cultured cells. Subsequent analysis of the CVS cultures and fetal tissues showed only normal female cells. Other false‐positive predictions involving non‐mosaic aneuploidies ( n = 13) were observed in the direct or culture method, but these cases involved rare aneuploidies: four cases of tetraploidy, two cases of trisomy 7, and one case each of trisomies 3, 8, 11, 15, 16,20, and 22. This indicates that rare aneuploidies observed in the direct or culture method should be subjected to follow‐up by amniocentesis. Two cases of unbalanced structural abnormalities detected in the direct method were not confirmed in cultured CVS or amniotic fluid. In addition, one structural rearrangement was misinterpreted as unbalanced from the direct method, leading to pregnancy termination prior to results from cultured cells showing a balanced, inherited translocation. False‐negative results ( n = 8) were observed only in the direct method, including one non‐mosaic fetal abnormality (trisomy 18) detected by the culture method and seven cases of fetal mosaicism (all detected by the culture method). Mosaicism was observed in 0.8 per cent of all cases, while pseudomosaicism (including single trisomic cells) was observed in 1.6 per cent of cases. Mosaicism was observed with equal frequency in the direct and culture methods, but was confirmed as fetal mosaicism more often in cases from the culture method (24 per cent) than in cases from the direct method (10 per cent). The overall rate of maternal cell contamination was 1.8 per cent for the culture method, but there was only one case of incorrect sex prediction due to complete maternal cell contamination which resulted in the birth of a normal male. The rate of maternal cell contamination was significantly higher in samples obtained by the transcervical sampling method (2. 16 per cent) than in samples obtained by the transabdominal method (0.79 per cent). From these data, it is clear that the culture method has a higher degree of diagnostic accuracy than the direct method, which should not be used as the sole diagnostic technique. The direct method can be a useful adjunct to the culture method, in which maternal cell contamination can lead to incorrect sex prediction and potentially to false‐negative diagnostic results.

About 2 per cent of specimens from chorionic villus sampling (CVS) analysed either on direct preparation of cytotrophoblast cells or after culture of mesenchymal stroma reveal confined placental mosaicism (CPM), most commonly involving chromosomal trisomy. A significantly higher rate of prenatal loss (22 per cent) as well as the presence of intrauterine growth retardation (IUGR) has been reported among pregnancies with CPM. To evaluate more precisely the effect of these aneuploid cell lines confined to the placenta on intrauterine fetal growth and fetal survival, we have studied 34 term placentae from pregnancies with CPM diagnosed on CVS and confirmed identical mosaicism in 17 of these placentae. There was a direct correlation between a high number of aneuploid cells present at CVS and a high likelihood of their detection in term placenta. Also, the proportion of aneuploid cells in the mosaic term placentae correlated with that observed in CVS specimens. Among 17 gestations with confirmed CPM at delivery, there were six cases of IUGR identified, five in liveborns and one associated with intrauterine death.

Of over 1000 patients referred to the Genetics & IVF Institute for chorionic villus sampling, 10% had an abnormal gestational sac at the time of initial presentation. Villus sampling could not be performed in 0.6% of cases, usually because of fibroids. Chorioamnionitis, probably related to the procedure, occurred in two patients (0.2%). The total fetal loss was 2.4%, which is not significantly different from the background fetal loss rate reported in normal pregnancies after eight weeks of gestation. The procedure-related loss was estimated as approximately 0.6%. The cytogenetic analysis routinely used was "direct" trophoblast karyotyping. There were no false-negative diagnoses. Trophoblastic mosaicism was observed in 1.3% of samples. Amniocentesis was performed in 3.5% of cases because of chorionic villus sampling failure, chorionic mosaicism, or elevated maternal serum alpha-fetoprotein (MSAFP) concentrations. Chorionic villus sampling was not associated with falsely elevated MSAFP levels at 15-18 weeks' gestation. Chorionic villus sampling has proved a safe and accurate prenatal diagnostic method associated with relatively low risk when performed by an experienced team, although additional amniocentesis will be necessary in a small percentage of pregnancies.