Several genetic variants in the CYP21A2 gene eliminate or reduce P450c21 activity, which is involved in cortisol biosynthesis. These variants can cause 21-hydroxylase deficient classical or nonclassical CAH, which are associated with several clinical symptoms including decreased fertility in males and females. The purpose of this study was to measure the carrier rates of genetic variants in CYP21A2 in fertility patients to determine if testing for these variants should be considered in this population. Retrospective. The Illumina Infinium HD Custom Genotyping platform was used to test for variants in the CYP21A2 gene, including c.293-13C>G (rs6467) and p.G425S (rs72552758) associated with classical CAH and p.H63L (rs9378252) and p.P454S (rs6445) associated with nonclassical CAH. Genotype frequencies were calculated based on data obtained from 2,188 clinical referrals from fertility centers. Documented informed consent to utilize clinical data in a de-identified manner was obtained. We found that 1,908 individuals (87.2%) do not carry any of the tested CYP21A2 variants. A total of 237 (10.83%) and 22 (1.01%) of the tested individuals were carriers or homozygous for the p.H63L nonclassical variant, respectively. Only one individual (0.05%) was homozygous for the p.P454S nonclassical variant. We identified 18 individuals (0.82%) that carried both nonclassical CAH variants. Further, two individuals (0.09%) carried both the classical c.293-13C>G and nonclassical p.H63L variants. Compared to findings from other studies, the nonclassical p.H63L variant is much more common in our population of fertility patients than previously reported. As of 2013, only 31 cases of p.H63L had been identified in individuals presenting with CAH [1]. However, we found that 12.76% of our population carries at least one copy of this variant. The literature suggests that the p.H63L variant reduces P450c21 activity and is associated with mild clinical features. The relatively high frequency of this variant in our population of fertility patients suggests the need to investigate the association of this variant with infertility specifically. If an association exists, this could inform hormone-based fertility treatment decisions.

ObjectiveTo evaluate the efficacy of Karyomapping, for PGD of complex SGDs.DesignPGD was carried out for 7 complex SGDs through utilization of Karyomapping (Kmapp). Cases were defined as 'complex' in occasions were embryos had to be tested for 2 different SGDs, HLA matching combined with PGD for SGD was needed, when consanguineous couples and couples with de novo mutations/no available family members for phase determination.Materials and MethodsOne biopsy was performed for 33 blastocysts. Each biopsied sample was WGA and aliquots of the products were used to perform Kmapp analysis in parallel with direct mutation detection ( PCR). 10 different SGDs and 1 HLA matching case were processed. Sibling embryos or isolated single sperm were assessed for phase determination in cases where no family members were available and/or de novo mutations.ResultsPreparation of the Kmapp/direct mutation test for each one of these cases was rapid and did not differ from time needed for preparation of non-complex SGD with the same method. Regarding the blastocysts tested, diagnosis was provided for all samples that were successfully amplified (29/33). Kmapp analysis was concordant with direct mutation detection in all samples tested. Utilisation of sibling embryos and single sperm was found to be highly successful for determination of phase on Kmapp. Also, Kmapp was able to give diagnosis for the embryos of all consanguineous cases processed, even though the number of informative, key, single nucleotide polymorphisms (SNPs) was considerably low. In total, 9/29 samples were found to be suitable for transfer.ConclusionKmapp utilizes a universal protocol applicable to every patient, to provide linkage-based PGD. Through this study this technology was proven to be highly efficient in providing a diagnosis in complex PGD cases, without increasing patient's waiting time for initiation of an IVF cycle. It is advisable that at least 2 sibling embryos/single sperms are processed with Kmapp for establishment of phase and that direct mutation detection is always performed in parallel for consanguineous cases. Karyomapping is expected to make PGD more accessible to patients under the 'complex cases' category. ObjectiveTo evaluate the efficacy of Karyomapping, for PGD of complex SGDs. To evaluate the efficacy of Karyomapping, for PGD of complex SGDs. DesignPGD was carried out for 7 complex SGDs through utilization of Karyomapping (Kmapp). Cases were defined as 'complex' in occasions were embryos had to be tested for 2 different SGDs, HLA matching combined with PGD for SGD was needed, when consanguineous couples and couples with de novo mutations/no available family members for phase determination. PGD was carried out for 7 complex SGDs through utilization of Karyomapping (Kmapp). Cases were defined as 'complex' in occasions were embryos had to be tested for 2 different SGDs, HLA matching combined with PGD for SGD was needed, when consanguineous couples and couples with de novo mutations/no available family members for phase determination. Materials and MethodsOne biopsy was performed for 33 blastocysts. Each biopsied sample was WGA and aliquots of the products were used to perform Kmapp analysis in parallel with direct mutation detection ( PCR). 10 different SGDs and 1 HLA matching case were processed. Sibling embryos or isolated single sperm were assessed for phase determination in cases where no family members were available and/or de novo mutations. One biopsy was performed for 33 blastocysts. Each biopsied sample was WGA and aliquots of the products were used to perform Kmapp analysis in parallel with direct mutation detection ( PCR). 10 different SGDs and 1 HLA matching case were processed. Sibling embryos or isolated single sperm were assessed for phase determination in cases where no family members were available and/or de novo mutations. ResultsPreparation of the Kmapp/direct mutation test for each one of these cases was rapid and did not differ from time needed for preparation of non-complex SGD with the same method. Regarding the blastocysts tested, diagnosis was provided for all samples that were successfully amplified (29/33). Kmapp analysis was concordant with direct mutation detection in all samples tested. Utilisation of sibling embryos and single sperm was found to be highly successful for determination of phase on Kmapp. Also, Kmapp was able to give diagnosis for the embryos of all consanguineous cases processed, even though the number of informative, key, single nucleotide polymorphisms (SNPs) was considerably low. In total, 9/29 samples were found to be suitable for transfer. Preparation of the Kmapp/direct mutation test for each one of these cases was rapid and did not differ from time needed for preparation of non-complex SGD with the same method. Regarding the blastocysts tested, diagnosis was provided for all samples that were successfully amplified (29/33). Kmapp analysis was concordant with direct mutation detection in all samples tested. Utilisation of sibling embryos and single sperm was found to be highly successful for determination of phase on Kmapp. Also, Kmapp was able to give diagnosis for the embryos of all consanguineous cases processed, even though the number of informative, key, single nucleotide polymorphisms (SNPs) was considerably low. In total, 9/29 samples were found to be suitable for transfer. ConclusionKmapp utilizes a universal protocol applicable to every patient, to provide linkage-based PGD. Through this study this technology was proven to be highly efficient in providing a diagnosis in complex PGD cases, without increasing patient's waiting time for initiation of an IVF cycle. It is advisable that at least 2 sibling embryos/single sperms are processed with Kmapp for establishment of phase and that direct mutation detection is always performed in parallel for consanguineous cases. Karyomapping is expected to make PGD more accessible to patients under the 'complex cases' category. Kmapp utilizes a universal protocol applicable to every patient, to provide linkage-based PGD. Through this study this technology was proven to be highly efficient in providing a diagnosis in complex PGD cases, without increasing patient's waiting time for initiation of an IVF cycle. It is advisable that at least 2 sibling embryos/single sperms are processed with Kmapp for establishment of phase and that direct mutation detection is always performed in parallel for consanguineous cases. Karyomapping is expected to make PGD more accessible to patients under the 'complex cases' category.

Carrier screening for recessive genetic diseases is offered to patients at fertility centers. Couples carrying the same genetic disease have a 25% chance of having an affected child. Such couples have several reproductive options available, including PGD. Our goal was to determine how many such couples pursue PGD. Retrospective. The Illumina Infinium HD Custom Genotyping platform was used to identify 1578 mutations associated with 190 high impact recessive genetic diseases. This analysis includes data obtained from 762 couples referred by fertility centers. Informed consent to utilize data in a de-identified manner was obtained. Carrier couples were identified. Couples that then pursued PGD through our partner laboratory were identified, and their PGD testing outcomes were reviewed. Of the 762 couples, 18 (2.4%) were identified as carriers of the same genetic disease as shown. Of these, 7 couples pursued single gene PGD through our partner laboratory.Table 1Disease# Carrier Samples# PGD SamplesPGD Status/ResultsSickle Cell Disease30Cystic Fibrosis42Preparation Complete, Preparation CompleteNonsyndromic Hearing Loss & Deafness: DFNB1 Related31InitiatedCarnitine Palmitoyltransferase II Deficiency10Familial Dysautonomia11Unaffected Embryos TransferredNiemann-Pick Disease: Type A10Hurler Syndrome11Unaffected/Non-Carrier Polar Bodies SelectedSpinal Muscular Atrophy: SMN1 Linked21InitiatedFamilial Mediterranean Fever10Tay-Sachs Disease11No Unaffected/Chromosomally Normal Embryos AvailableTotal187 Open table in a new tab Results show that 11/18 carrier couples referred by fertility centers chose not to pursue PGD. There are several explanations for this. PGD may not be consistent with a couple's personal beliefs or may not have the financial resources to pursue PGD. Additionally, it is possible that some couples pursued PGD through another laboratory, became pregnant without fertility treatment, or chose not to pursue fertility treatment in general. In this study, at least one affected embryo was detected for each couple that pursued and completed PGD testing, demonstrating the importance of performing PGD testing in these cases.

It is well established that chromosomes occupy distinct positions within the interphase nuclei, conferring a potential functional implication to the genome. In addition, alterations in the nuclear organisation patterns have been associated with disease phenotypes (e.g. cancer or laminopathies). The human sperm is the smallest cell in the body with specific DNA packaging and the mission of delivering the paternal genome to the oocyte during fertilisation. Studies of nuclear organisation in the sperm have postulated nonrandom chromosome position and have proposed a chromocentre model with the centromeres facing toward the interior and the telomeres toward the periphery of the nucleus. Most studies have assessed the nuclear address in the sperm longitudinally predominantly using centromeric or telomeric probes and to a lesser extent with whole chromosome paints. To date, studies investigating the radial organisation of human sperm have been limited. The purpose of this study was to utilise whole chromosome paints for six clinically important chromosomes (18, 19, 21, 22, X, and Y) to investigate nuclear address by assessing their radial and longitudinal nuclear organisation. A total of 10,800 sperm were analysed in nine normozoospermic individuals. The results have shown nonrandom chromosome position for all chromosomes using both methods of analysis. We present novel radial and polar analysis of chromosome territory localization within the human sperm nucleus. Specifically, a hierarchical organisation was observed radially with chromosomes organised from the interior to the periphery (chromosomes 22, 21, Y, X, 19, and 18 respectively) and polar organisation from the sperm head to tail (chromosomes X, 19, Y, 22, 21, and 18, respectively). We provide evidence of defined nuclear organisation in the human sperm and discuss the function of organisation and potential possible clinical ramifications of these results in regards to male infertility and early human development.

Commercially obtained Buffalo rat liver (BRL) cells were grown in monolayer culture. The effect of BRL cell co-culture with assisted hatching on embryo development, implantation and pregnancy was investigated in a population of 200 'first-time' in-vitro fertilization (IVF) patients, subdivided into three groups according to the methods of fertilization [IVF; intracytoplasmic sperm injection (ICSI); ICSI/IVF]. Assisted hatching was performed on all embryos chosen for transfer. Following co-culture, the overall embryo quality, implantation rate and pregnancy rates were not significantly different from the controls. However, when grouped according to fertilization method, co-culture was found to have an impact on pregnancy and implantation rates in the group undergoing conventional IVF. Using co-culture with assisted hatching, we were able to achieve a 58% (38/65) clinical pregnancy rate with a 49% (32/65) live birth rate and a 26% (60/235) implantation rate. No changes in the pregnancy and implantation rates were apparent in ICSI or ICSI/IVF subgroups. This is the first prospective, randomly controlled study which reports the use of BRL cell co-culture for human IVF for a large number of patients undergoing IVF for the first time.

Videocinematography and image analysis procedures were utilized to evaluate the effect of conventional and coculture methodologies on morphological parameters in human embryos derived from in-vitro fertilization (IVF). Following 24-30 h of in-vitro development, cocultured embryos had more acceptable morphological features and less fragmentation present than embryos cultured in medium alone. Cocultured embryos were more advanced at the time of replacement when compared with conventionally cultured embryos. Zona pellucida variation (> or = 20%) also occurred more frequently in cocultured embryos. The morphological characteristic most enhanced after coculture was blastomere expansion. Patients who became pregnant across both culture treatments had a higher proportion of morphologically normal embryos replaced than patients who failed to achieve an ongoing pregnancy. Clinical pregnancy rate for patients following coculture was 49%, which was greater (P < 0.05) than the 29% detected for patients with embryos in the conventional culture group.

Videocinematography and image analysis procedures were utilized to evaluate the effect of conventional and coculture methodologies on morphological parameters in human embryos derived from in-vitro fertilization (IVF). Following 24-30 h of in-vitro development, cocultured embryos had more acceptable morphological features and less fragmentation present than embryos cultured in medium alone. Cocultured embryos were more advanced at the time of replacement when compared with conventionally cultured embryos. Zona pellucida variation (> or = 20%) also occurred more frequently in cocultured embryos. The morphological characteristic most enhanced after coculture was blastomere expansion. Patients who became pregnant across both culture treatments had a higher proportion of morphologically normal embryos replaced than patients who failed to achieve an ongoing pregnancy. Clinical pregnancy rate for patients following coculture was 49%, which was greater (P < 0.05) than the 29% detected for patients with embryos in the conventional culture group.