Article
Potential selection of genetically balanced spermatozoa based on the hypo-osmotic swelling test in chromosomal rearrangement carriers

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Abstract

Chromosomal translocations and other balanced rearrangements, although usually associated with a normal phenotype, can lead to the transmission of an abnormal unbalanced genome to the offspring. Balanced and unbalanced spermatozoa, being indistinguishable, cannot be selected or deselected for prior to IVF and pre-implantation genetic diagnosis. Spermatozoa from 16 chromosomal rearrangement carriers were studied. After incubation in a hypo-osmotic solution (hypo-osmotic swelling test, or HOST), spermatozoa were fixed on microscope slides. The chromosomally balanced or unbalanced status corresponding to each observed class of flagellar conformation was evaluated through fluorescent in-situ hybridization (FISH). We show here a specific type of spermatozoa, with a distinct flagellar conformation that was associated with a balanced genetic content. HOST is a simple, low-cost and time-honoured procedure initially developed to distinguish immotile viable from non-viable spermatozoa. We demonstrate that it can also be used to identify genetically balanced spermatozoa in chromosomal rearrangement carriers, with a 96% decrease in the proportion of unbalanced spermatozoa after selection. This may potentially improve reproductive prognosis in affected couples if used prior to pre-implantation genetic diagnosis (PGD), and clinical utility and efficacy should be evaluated in further studies.

Introduction

Chromosomal rearrangements, such as translocations and inversions, exist with a prevalence of 1/500 in the general population. In most cases, they are balanced and asymptomatic but are nevertheless associated with a risk of transmitting an unbalanced karyotype to the offspring. Indeed, in these patients, a variable proportion of gametes are genetically unbalanced and are associated with different adverse outcomes such as spontaneous abortions, pregnancy terminations, fetal malformations, or learning disabilities in resulting children (McKinlay Gardner and Sutherland, 2011). Genetically unbalanced spermatozoa are morphologically indistinguishable from their balanced counterparts. Affected couples are therefore advised to either conceive naturally, with planned prenatal diagnosis, or to seek pre-implantation genetic diagnosis (PGD) (Hirshfeld-Cytron et al., 2011). However, the former option implies a pregnancy termination in the case of an adverse result, while the latter is not available in every country and leads to the loss of oocytes fertilized with chromosomally unbalanced spermatozoa.

It has been previously described that discontinuous gradient centrifugation increases the rate of balanced spermatozoa in structural chromosomal rearrangement carriers (Rouen et al., 2013a). However, selection is only partial, with a mean decrease in abnormal spermatozoa of around 30%, which led us to seek a more discriminating procedure, which furthermore needed to be compatible with subsequent assisted reproduction techniques. We postulated that the hypo-osmotic swelling test (HOST) could be used for this purpose. This procedure has been used for several decades to differentiate viable from non-viable spermatozoa, particularly in patients with akinetozoospermia (Jeyendran et al, 1984, World Health Organization, 2010). In the last few years, several authors have shown a correlation between sperm morphology after HOST and several parameters such as DNA fragmentation, membrane integrity, protamine deficiency and aneuploidy (Bassiri et al, 2012, Pang et al, 2010, Stanger et al, 2010, Zeyneloglu et al, 2000).

We therefore postulated that the flagellar conformation after incubation in a hypo-osmotic solution could be correlated with chromosomal content in spermatozoa from structural chromosomal rearrangement carriers.

Section snippets

Materials and methods

Fifteen chromosomal translocation carriers (10 reciprocal translocations and five Robertsonian translocations) as well as one pericentric inversion carrier were studied. Additionally, five normal controls were included for comparison of the proportion of the different HOST classes between them and the rearrangement carriers. These controls took part in IVF cycles for female indication. Most of these patients were initially consulted for infertility, recurrent spontaneous abortions or fetal

Results

When incubated in a hypo-osmotic solution, spermatozoa from translocation carriers underwent morphological modifications of their flagella, just as spermatozoa from patients with a normal karyotype do. As a whole, these modifications were similar to those originally reported by Jeyendran et al. (1984) and included in the WHO document (World Health Organization, 2010), and allowed us to classify sperm cells in one of the HOST A to G classes (Figure 2).

Because the FISH analysis was not conducted

Discussion

It has been previously demonstrated that in chromosomal rearrangement carriers, unbalanced spermatozoa have a higher level of apoptosis and DNA fragmentation (Brugnon et al, 2010, Rouen et al, 2013a), that their nuclei are less dense (Rouen et al., 2013b) and significantly larger (Rouen et al., 2015). We suggest that the fine spermatic nuclear architecture may be disrupted by chromosomal imbalances, and that this may hinder the normal condensation of the nucleus. Indeed, spermatic DNA is

Alexandre Rouen is a medical geneticist at Trousseau Hospital, Paris, France. He specializes in infertility of genetic origin, and conducts research focusing on improving reproductive care in patients with genetic defects, notably chromosomal rearrangements.

Key message

In male chromosomal rearrangement carriers, sperm selection based on the hypo-osmotic swelling test could potentially be used to select for genetically balanced spermatozoa, prior to pre-implantation genetic

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    Alexandre Rouen is a medical geneticist at Trousseau Hospital, Paris, France. He specializes in infertility of genetic origin, and conducts research focusing on improving reproductive care in patients with genetic defects, notably chromosomal rearrangements.

    Key message

    In male chromosomal rearrangement carriers, sperm selection based on the hypo-osmotic swelling test could potentially be used to select for genetically balanced spermatozoa, prior to pre-implantation genetic diagnosis, in order to improve reproductive prognosis in affected couples.

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