Meiotic recombination between highly-similar duplicated sequences (non-allelic homologous recombination, NAHR) generates deletions, duplications, inversions, and translocations, and is responsible for genetic diseases known as 'genomic disorders', most of which are caused by altered copy number of dosage sensitive genes. NAHR Hotspots have been identified within some duplicated sequences. We have developed sperm-based assays to measure the de novo rate of reciprocal deletions and duplications at 4 NAHR hotspots. We used these assays to dissect the relative rates of NAHR between different pairs of duplicated sequences. We show that: (i) these NAHR hotspots are specific to meiosis, (ii) deletions are generated at a higher rate than their reciprocal duplications in the male germline and (iii) some of these genomic disorders are likely to have been under-ascertained clinically, most notably the duplication of 7q11, the reciprocal of the Williams-Beuren Syndrome deletion.Genomic disorders are diseases caused by recurrent meiotic chromosomal rearrangements involving unstable genomic architectures 1. Most frequently these involve non-allelic homologous recombination (NAHR) between highly similar duplicated sequences. NAHR between duplicated sequences in direct orientation, results in deletion and duplication of intervening sequences, whereas inversions result from NAHR between duplicated sequences in inverted orientation. The predominant pathogenic mechanism for the genomic disorders associated with deletions and duplications is altered copy number of dosage sensitive genes 2. In addition to its role in genomic disorders, NAHR is one of the major mechanisms contributing to non-pathogenic structural variation in the human genome 3. The breakpoints of rearrangements caused by NAHR have been shown to cluster in defined hotspots within duplicated sequences, in a manner akin to that of allelic recombination hotspots 4.A simple model of NAHR suggests that recombination between duplicated sequences (paralogues) can take place in one of three ways, between paralogues on the same chromatid, on sister chromatids or on the homologous chromosome (Figure 1). In only the latter two cases are deletion and duplication reciprocal products of NAHR. According to this model of NAHR, the relative rates of deletion and duplication will be determined by the relative contribution that intra-chromatid NAHR makes to the overall frequency of meiotic NAHR, and the rate of duplication (β+γ) should never exceed the rate of deletion (α+β+γ). To address this question, by comparing directly rates of deletion and duplication, we developed breakpoint-specific real-time PCR assays to measure NAHR activity in the male germline at 4 known hotspots. These hotspots lie within: the WBS-LCRs (Low Copy Repeats) that sponsor the WBS deletion and dup7(q11.23) duplication 10; the AZFa-HERVs (Human Endogenous RetroVirus) that sponsor the AZFa deletion that causes male infertility and its reciprocal, apparently asymptomatic, duplication 11,12; the CMT1A-REPs...

A major puzzle in biology is how mammalian sperm maintain the correct swimming direction during various phases of the sexual reproduction process. Whilst chemotaxis may dominate near the ovum, it is unclear which cues guide spermatozoa on their long journey towards the egg. Hypothesized mechanisms range from peristaltic pumping to temperature sensing and response to fluid flow variations (rheotaxis), but little is known quantitatively about them. We report the first quantitative study of mammalian sperm rheotaxis, using microfluidic devices to investigate systematically swimming of human and bull sperm over a range of physiologically relevant shear rates and viscosities. Our measurements show that the interplay of fluid shear, steric surface-interactions, and chirality of the flagellar beat leads to stable upstream spiralling motion of sperm cells, thus providing a generic and robust rectification mechanism to support mammalian fertilisation. A minimal mathematical model is presented that accounts quantitatively for the experimental observations.DOI: http://dx.doi.org/10.7554/eLife.02403.001

Aneuploidy in human eggs is the leading cause of pregnancy loss and several genetic disorders such as Down's syndrome. Most aneuploidy results from chromosome segregation errors during the meiotic divisions of an oocyte, the egg's progenitor cell. The basis for particularly error-prone chromosome segregation in human oocytes is not known. Here we analyzed meiosis in over 100 live human oocytes and identified an error-prone chromosome-mediated spindle assembly mechanism as major contributor to chromosome segregation defects. Human oocytes assembled a meiotic spindle independently of either centrosomes or other microtubule organizing centers. Instead, spindle assembly was mediated by chromosomes and the small GTPase Ran in a process requiring ~16 hours. This unusually long spindle assembly period was marked by intrinsic spindle instability and abnormal kinetochore-microtubule attachments, which favor chromosome segregation errors and provide a possible explanation for high rates of aneuploidy in human eggs.Meiosis in human oocytes is more prone to chromosome segregation errors than mitosis (1, 2), meiosis during spermatogenesis (3, 4) and female meiosis in other organisms (3,5). Despite its importance for fertility and human development, meiosis in human eggs has hardly been studied. Human oocytes are only available in small numbers, warranting singlecell assays capable of extracting maximal information. While high resolution-live cell microscopy is an ideal method, oocyte development in the ovary poses challenges to direct imaging. We therefore established an experimental system (6) for ex vivo high resolution fluorescence microscopy of human oocytes freshly harvested from women undergoing gonadotropin-stimulated in vitro fertilization cycles. To establish the major stages of meiosis in this system, we simultaneously monitored microtubules and chromosomes for ~24-48 hours ( Fig. 1 and movie S1). Similar to the situation in situ (7), human oocytes matured into fertilizable eggs over this time course as judged by the formation of a polar body. The morphologically identifiable stages (Fig. 1A) Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts breakdown (NEBD, set to 0 hours) provided a time-resolved framework for human oocyte meiosis (Fig. 1B). This reference timeline post NEBD is used throughout this paper.Before NEBD, chromosomes were highly condensed and clustered around the nucleolus. Instead of rapidly nucleating microtubules upon NEBD, human oocytes first formed a chromosome aggregate that was largely devoid of microtubules (Fig. 1A, movie S1 and fig. S1, A and B). Microtubules were first observed at ~5 hours, when they started to form a small aster within the chromosome aggregate. As the microtubule aster grew, the chromosomes became individualized and oriented on the surface of the aster with their kinetochores facing inwards. The microtubule aster then extended into an early bipolar spindle that carried the chromosomes on its surface (Fig. 1A, movie S1 and fig. S1, C to E)...

Chromosome errors, or aneuploidy, affect an exceptionally high number of human conceptions, causing pregnancy loss and congenital disorders. Here, we have followed chromosome segregation in human oocytes from females aged 9 to 43 years and report that aneuploidy follows a U-curve. Specific segregation error types show different age dependencies, providing a quantitative explanation for the U-curve. Whole-chromosome nondisjunction events are preferentially associated with increased aneuploidy in young girls, whereas centromeric and more extensive cohesion loss limit fertility as women age. Our findings suggest that chromosomal errors originating in oocytes determine the curve of natural fertility in humans.

Chromosome segregation errors occurring during the meiotic divisions of a human oocyte are the leading cause of pregnancy loss and several genetic disorders. When chromosomes fail to split into perfect halves during meiosis, the embryo cannot survive or will have a genetic defect, such as Down syndrome. Despite the importance of meiosis in human eggs for fertility and human development, the basis for error-prone chromosome segregation is not known.The authors developed an experimental system for ex vivo high-resolution fluorescence microscopy that allowed them to examine human oocytes freshly harvested from women undergoing gonadotropin-stimulated, in vitro fertilization cycles. Through examination of meiosis in more than 100 live human oocytes, an error-prone, chromosome-mediated, spindle assembly mechanism was identified as a major contributor to chromosome segregation defects. Human oocyte spindle assembly was mediated primarily by chromosomes and the small guanosine triphosphatase Ran independent of centrosomes or other microtubule organizing centers in a process requiring about 16 hours. This unusually slow process is in sharp contrast to mitotic spindles and meiotic spindles in mouse oocytes and other species, which rarely become unstable upon establishment of a bipolar spindle, thus rendering meiosis more efficient and less prone to segregation errors.Spindles assembled during meiosis in human oocytes display a high proportion of abnormal kinetochore-microtubule attachments and are intrinsically unstable. Progression into anaphase with abnormal attachments put human oocytes at risk of chromosome segregation errors, providing 1 mechanism for the high rates of aneuploidy.

Aneuploidy in human eggs is the leading cause of pregnancy loss and Down’s syndrome. Aneuploid eggs result from chromosome segregation errors when an egg develops from a progenitor cell, called an oocyte. The mechanisms that lead to an increase in aneuploidy with advanced maternal age are largely unclear. Here, we show that many sister kinetochores in human oocytes are separated and do not behave as a single functional unit during the first meiotic division. Having separated sister kinetochores allowed bivalents to rotate by 90 degrees on the spindle and increased the risk of merotelic kinetochore-microtubule attachments. Advanced maternal age led to an increase in sister kinetochore separation, rotated bivalents and merotelic attachments. Chromosome arm cohesion was weakened, and the fraction of bivalents that precociously dissociated into univalents was increased. Together, our data reveal multiple age-related changes in chromosome architecture that could explain why oocyte aneuploidy increases with advanced maternal age.DOI: http://dx.doi.org/10.7554/eLife.11389.001

There is considerable evidence to suggest that polypeptide growth factors from either the oviduct or the endometrium can control preimplantation development of the mammalian embryo. These act directly through receptors expressed on the embryo. In addition, embryos also produce growth factors. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the pattern of expression of mRNAs encoding several growth factor ligand and receptor genes throughout preimplantation development of cryopreserved human embryos. Transcripts encoding the receptor for c-fms, the receptor for colony-stimulating factor-1 (CSF-1), and c-kit (the receptor for stem cell factor [SCF]) were expressed throughout preimplantation development. Other growth factor ligand and receptor transcripts were expressed in a stage-specific manner: these included receptors for interleukin (IL)-6 (IL-6R), leukemia inhibitory factor (LIFR), tumor necrosis factor alpha (TNF alpha) (TNFRp80 and TNFRp60), and gp130. The transcripts for gp130 and the ligand SCF showed stage-specific splice variants. Blastocysts expressed a novel cDNA encoding gp130, which predicts a truncated form lacking the intracellular signaling domain. No expression of mRNAs encoding LIF, CSF-1, or the cloned receptor for platelet-activating factor was seen in any embryonic stage studied. We have shown that RT-PCR provides a sensitive and powerful method for identifying transcripts encoding growth factors and their receptors in single human embryos. The method is economical, allowing the expression pattern of many genes to be determined from a single embryo. These data are important in defining which cytokines may be involved in regulating human preimplantation development and when they may act.

The improved survival in recent years of young males suffering from cancer, and an understanding of the gonadotoxic effects of chemotherapy treatment, have motivated patients and clinicians to preserve fertility potential before embarking on adjuvant therapy. Among 231 men (mean age 28.0; range 15-56 years) diagnosed with malignant disease and referred to our unit for semen cryopreservation, 112 patients (49.8%) had reduced sperm quality of <10 x 10(6) motile spermatozoa per ejaculate; however, most had sufficient suitable spermatozoa for freezing. In 40 patients (17.3 %) the semen samples were not frozen because of complete azoospermia (n = 32) or only immotile sperm in the ejaculate (n = 2), while six men were unable to produce a single sample. Some 79 men had testicular tumours (group I), 121 suffered from haematological malignancy (leukaemia or lymphoma; group II), and 27 had cancer of different causes (group III). Men in group I had significantly lower (P < 0.001) sperm quality compared with groups II and III. There was no difference between patients with seminoma and non-seminoma tumours. In the haematological malignancy group there was no difference in sperm parameters between leukaemia (n = 12) and lymphoma (n = 77) patients, but patients with Hodgkin's lymphoma had significantly lower sperm quality compared with non-Hodgkin's lymphoma. Following chemotherapy, six couples attended the clinic for assisted conception treatment using the frozen semen. Two had successful intrauterine insemination cycles which each resulted in delivery of a healthy girl; one couple had conceived in their first in-vitro fertilization (IVF) attempt, followed by delivery of healthy twins. Two women conceived after intracytoplasmic sperm injection treatment and the sixth woman achieved only biochemical pregnancy after numerous IVF and frozen embryo replacement cycles. We recommend that a properly designed programme for semen cryopreservation for cancer patients should be developed in leading tertiary assisted conception centres, which have adequate facilities and experience for cryopreservation and can offer the whole range of appropriate assisted reproductive treatment and counselling.