Martine Albert

Can intracytoplasmic morphologically selected sperm injection be used to select normal-sized sperm heads in infertile patients with macrocephalic sperm head syndrome?

OBJECTIVE To study the chromosomal content of spermatozoa selected by intracytoplasmic morphologically selected sperm injection (IMSI) in cases of macrocephalic sperm head syndrome. DESIGN Case report. SETTING Obstetrics, gynecology, urology, and reproductive biology departments. PATIENT(S) Two infertile patients with large-headed spermatozoa. INTERVENTION(S) Fluorescence in situ hybridization on selected spermatozoa with normal-sized heads after IMSI selection. MAIN OUTCOME MEASURE(S) Percentages of polyploid, diploid, haploid aneuploid, and normal spermatozoa. RESULT(S) Of the six spermatozoa that could be selected, all were haploid but aneuploid. CONCLUSION(S) Absence of normal haploid spermatozoa among high magnification-selected spermatozoa contraindicated IMSI for these two patients.

Cryopreservation of Human Spermatozoa Decreases the Number of Motile Normal Spermatozoa, Induces Nuclear Vacuolization and Chromatin Decondensation

Even though cryopreservation of human spermatozoa is known to alter sperm motility and viability, it may also induce nuclear damages. The present study set out to determine whether or not cryopreservation alters motile sperm morphology under high magnification and/or is associated with chromatin decondensation. For 25 infertile men, we used high-magnification microscopy to determine the proportions of various types of motile spermatozoa before and after freezing-thawing: morphometrically normal spermatozoa with no vacuole (grade I), ≤ 2 small vacuoles (grade II), at least 1 large vacuole or >2 small vacuoles (grade III), and morphometrically abnormal spermatozoa (grade IV). The spermatozoas chromatin condensation and viability were also assessed before and after freezing-thawing. Cryopreservation induced sperm nuclear vacuolization. It decreased the proportion of grade I + II spermatozoa (P < .001). It induced a decrease in the sperm viability rate (P < .001) and increased the proportion of sperm with noncondensed chromatin (P < .001). The latter parameter was strongly correlated with sperm viability (r = 0.71; P < .001). However, even motile sperm presented a failure of chromatin condensation after freezing-thawing, because the proportion of sperm with noncondensed chromatin was correlated with high-magnification morphology (r = -0.49 and 0.49 for the proportions of grade I + II and grades III + IV, respectively; P < .001). Cryopreservation alters the organelle morphology of motile human spermatozoa and induces sperm chromatin decondensation. High-magnification microscopy may be useful for evaluating frozen-thawed spermatozoa before use in assisted reproductive technology procedures (such as intrauterine insemination, in vitro fertilization, and intracytoplasmic sperm injection) and for performing research on cryopreservation methods. If frozen-thawed sperm is to be used for intracytoplasmic sperm injection, morphological selection under high magnification may be of particular value.

Tumor necrosis factor-alpha −308 polymorphism in infertile men with altered sperm production or motility

BACKGROUND One of the most well-documented cytokines suspected as a hazard to male fertility is tumor necrosis factor-alpha (TNFalpha). Genetic factors such as single-nucleotide polymorphisms (SNPs) in the TNF gene cluster impact TNFalpha levels. Our objective was to establish the potential involvement of -308 TNF SNP in male infertility risk. METHODS In 684 infertile male patients undergoing an intracytoplasmic sperm injection procedure, we used allele-specific polymerase chain reaction (PCR) and PCR-RFLP to investigate the distribution of the guanine (G)-to-adenosine (A) substitution at position -308 in the promoter region of the TNFalpha gene. RESULTS An increased frequency of the -308 TNFalpha A allele was found in patients with low sperm count of testicular origin [P = 0.002; odds ratio (OR) = 2.93] or with normal production count but altered sperm motility (P = 0.003; OR = 2.32), compared with a patient group with normal sperm count and quality (morphology and motility). In patients with low sperm count exhibiting TNFalpha A allele, compared with those with G allele, an alteration in hormonal balance was observed with increased inhibin B levels and subsequent reduced FSH plasma levels, leading to an FSH/inhibin B ratio roughly half as high (from 0.07 +/- 0.01 in TNFA versus 0.13 +/- 0.02 in TNFG allele groups, P < 0.0001). CONCLUSION As the -308 TNFalpha A allele has been associated with an increased expression/production of TNFalpha, the potential use of therapies based on inhibition of TNFalpha activities could represent possible therapeutic opportunities for patients with low sperm count (i.e. primary testicular dysfunction) or with altered sperm motility.

The High Frequency of Sperm Aneuploidy in Klinefelter Patients and in Nonobstructive Azoospermia Is Due to Meiotic Errors in Euploid Spermatocytes

For nonobstructive azoospermic (NOA) patients with a normal karyotype or for Klinefelter syndrome (47,XXY) patients, intracytoplasmic sperm injection is associated with an increased aneuploidy risk in offspring. We examined testicular cells from patients with different azoospermia etiologies to determine the origin of the aneuploid spermatozoa. The incidence of chromosome abnormalities was investigated in all types of azoospermia. Four study subgroups were constituted: Klinefelter patients (group 1), NOA patients with spermatogenesis failure but a normal karyotype (group 2), obstructive azoospermic patients with normal spermatogenesis (group 3), and control patients with normal sperm (group 4). The pachytene stage (in the three azoospermic groups) and postmeiotic cells (in all groups) were analyzed with fluorescence in situ hybridization. No aneuploid pachytene spermatocytes were observed. Postmeiotic aneuploidy rates were higher in the two groups with spermatogenesis failure (5.3% and 4.0% for groups 1 and 2, respectively) than in patients with normal spermatogenesis (0.6% for group 3 and group 4). Whatever the etiology of the azoospermia, the spermatozoa originated from euploid pachytene spermatocytes. These results strengthen the hypothesis whereby sperm aneuploidy in both Klinefelter patients and NOA patients with a normal karyotype results from meiotic abnormalities and not from aneuploid spermatocytes. The fact that sperm aneuploidy was more frequent when spermatogenesis was altered suggests a deleterious testicular environment. The study results also provide arguments for offering preimplantation genetic diagnosis or prenatal diagnosis when a pregnancy occurs for fathers with NOA (whatever the karyotype).

A genome-wide DNA methylation study in azoospermia.

The objective of this study was to assess genome‐wide DNA methylation in testicular tissue from azoospermic patients. A total of 94 azoospermic patients were recruited and classified into three groups: 29 patients presented obstructive azoospermia (OA), 26 displayed non‐obstructive azoospermia (NOA) and successful retrieval of spermatozoa by testicular sperm extraction (TESE+) and 39 displayed NOA and failure to retrieve spermatozoa by TESE (TESE−). An Illumina Infinium Human Methylation27 BeadChip DNA methylation array was used to establish a testicular DNA methylation pattern for each type of azoospermic patient. The OA and NOA groups were compared in terms of the relative M‐value (the log2 ratio between methylated and non‐methylated probe intensities) for each CpG site. We observed significantly different DNA methylation profiles for the NOA and OA groups, with differences at over 9000 of the 27 578 CpG sites; 212 CpG sites had a relative M‐value >3. The results highlighted 14 testis‐specific genes. Patient clustering with respect to these 212 CpG sites corresponded closely to the clinical classification. The DNA methylation patterns showed that in the NOA group, 78 of the 212 CpG sites were hypomethylated and 134 were hypermethylated (relative to the OA group). On the basis of these DNA methylation profiles, azoospermic patients could be classified as OA or NOA by considering the 212 CpG sites with the greatest methylation differences. Furthermore, we identified genes that may provide insight into the mechanism of idiopathic NOA.

Gamete cytogenetic study in couples with implantation failure: aneuploidy rate is increased in both couple members

PurposeImplantation failure is known to be associated with an increased risk of aneuploidy in embryos, a situation leading to a pre-implantation genetic screening, not allowed in different countries like France. Our aim was to evaluate the gamete aneuploidy incidence in this context, using first polar body and spermatozoa aneuploidy screening.MethodsThree groups were considered: 11 couples with pregnancy obtained after IVF for female infertility (group 1); 20 couples with pregnancy obtained after IVF for male infertility (group 2); and 35 couples with implantation failure (group 3). In group 3, 28 couples treated by ICSI volunteered for first polar body analysis (PB1).ResultsSpermatozoa aneuploidy rate was increased in groups 2 (1.6%) and 3 (2.1%) in comparison to group 1 (0.6%). PB1 aneuploidy rate was 35.4% in group 3. Finally, eight couples (32%) had no particular chromosomal risk in gametes, 15/25 (60%) presented an increased spermatic (>2%) or oocyte (>1/3) aneuploidy rate, and 2/25 (8%) had both.ConclusionThose results confirm that implantation failure has a heterogeneous origin, that gamete chromosome abnormality rate is one of the major contributing factors, and that 1st Polar body and spermatozoa aneuploidy screening or pre-implantation genetics screening may be indicated for these couples.

Evaluation de l’apport de la méthode d’observation des spermatozoïdes à fort grossissement en ICSI

ResumeNous avons souhaité évaluer l’intérêt de la sélection des spermatozoïdes par la méthode d’observation à fort grossissement, appelée MSOME (Motile Sperm Organelle Morphology Examination), par rapport à celle habituellement réalisée en ICSI. Notre objectif était de déterminer le pourcentage de spermatozoïdes sélectionnés en ICSI conventionnelle qui s’avéraient anormaux à fort grossissement, et d’évaluer la valeur prédictive de ce paramètre sur l’issue de la tentative d’ICSI.L’étude a concerné 25 tentatives d’ICSI sans sélection d’indication. Pour chaque patient, 25 spermatozoïdes mobiles et “injectables” d’après l’évaluation morphologique conventionnelle en ICSI ont été observés en MSOME à un grossissement supérieur à ×4500 et classés selon une grille de lecture adaptée des travaux de Bartoov et de la classification de David modifiée. Nous avons comparé les résultats de MSOME et l’issue des ICSI correspondantes.Dans cette courte série d’ICSI sans sélection d’indication, nous avons retrouvé des fréquences élevées d’anomalies (supérieures à 70%) et notamment de vacuoles nucléaires. Nous n’avons pas retrouvé de valeur prédictive de la morphologie spermatique évaluée à fort grossissement, y compris des vacuoles, pour le taux de fécondation, la qualité embryonnaire ou l’issue de l’ICSI. De plus, des grossesses ont été obtenues avec des spermes très altérés. Dans cette série, les vacuoles ne semblent pas avoir de signification péjorative pour l’obtention d’une grossesse.Plusieurs questions sont soulevées à l’issue de ce travail : quel est le support et la signification des vacuoles observées en MSOME ? Y a-t-il une corrélation entre la morphologie spermatique et le contenu génétique ? Enfin, des études prospectives randomisées semblent nécessaires afin de valider les indications potentielles de cette technique.AbstractDue to the growing interest in the method of high-magnification sperm observation and selection proposed for the specific indication of ICSI failure, the authors evaluated the technique in unselected ICSI.The aim of this study was to evaluate the relevance of Motile Sperm Organelle Morphology Examination (MSOME) compared with usual selection performed in ICSI. In a series of conventionally selected sperm for ICSI, the number with an abnormal appearance on high magnification was determined and the predictive value of this parameter on ICSI outcome was assessed.The study included 25 successive unselected ICSI attempts in the IVF Laboratory of Poissy Hospital (France). ICSI were performed according to usual protocols used in the laboratory. Twenty five motile spermatozoa of the migrated fraction, still available after ICSI, and “injectable— according to conventional morphology assessment in ICSI (“normal” or “as normal as possible” with magnification of ×200–400) were assessed by MSOME (higher than ×4500) and classified according to criteria adapted from Bartoov’s work and taking into account David’s sperm morphology classification. We compared the results of MSOME and ICSI results.In this small series of ICSI with diverse indications, we found very high frequencies of abnormalities (more than 70%), particularly nuclear vacuoles. No predictive value of the morphology of sperm assessed with high magnification (including vacuoles) was found for fertilization rate, embryo quality and ICSI outcome. In contrast with previous reports, pregnancies were obtained with very abnormal sperms. In this series of unselected ICSI, nuclear vacuoles do not seem to have a pejorative impact on pregnancy outcome.This study raises several perspectives. It would be interesting to understand the “anatomical” basis for vacuoles observed with MSOME and their meaning. The question of the phenotype-genotype relation, i.e. the possible correlation between sperm morphology and genetic content could be investigated. Finally, a prospective analysis should be performed in clearly defined indications to validate the potential applications of the method for high-magnification sperm observation and selection.

Are zona pellucida laser drilling and polar body biopsy safe for in vitro matured oocytes

IntroductionPreconception diagnosis requires first polar body biopsy. When the hole in the zona pellucida is made with a laser beam, heat propagation could, like the biopsy itself, be deleterious. Our aim was to evaluate the effect of this technique on human in vitro matured oocyte and embryo development.MethodsOne hunded fifty five retrieved immature oocytes from 75 women, matured in vitro, were distributed in 3 groups: 50 oocytes in a control group, without laser drilling and first polar body biopsy, 52 oocytes in a group with only laser drilling, and 53 oocytes in a group with both laser drilling and first polar body biopsy. Safety was evaluated using four criteria: [1] oocyte lysis rate, [2] oocyte activation rate, [3] oocyte development after calcium ionophore treatment, [4] and embryo chromosome breakage incidence after Tarkowski preparation.ResultsNo difference in the four criteria was observed between the 3 oocyte groups.ConclusionsWe did not find evidence of deleterious effect of laser drilling and first polar body biopsy on in vitro matured oocytes, according to our criteria.

Status of the executioner step of apoptosis in human with normal spermatogenesis and azoospermia

OBJECTIVE To localize the different proteins involved in the executioner step of apoptosis in the human testicular tissue: effector caspases (3, 6, and 7), caspase inhibitors called inhibitor of apoptosis proteins (IAPs, such as XIAP), and IAP inhibitors such as Smac/DIABLO and to investigate XIAP and Smac expression and activation of caspase-3 in azoospermia. DESIGN Retrospective study. SETTING The Biology of Reproduction Center of Poissy and Inserm U407, France. PATIENT(S) Twenty-five patients diagnosed as azoospermic and 4 patients with normal testicular histology. INTERVENTION(S) Testicular biopsies for histopathological assessment. MAIN OUTCOME MEASURE(S) Localization of proteins by immunohistochemistry. RESULT(S) Effector caspase-7 seemed to be absent from normal human testes, whereas procaspase-3 and procaspase-6 were detected in both somatic and germ cells. XIAP was mainly expressed in Sertoli cells, whereas its inhibitor Smac was detected in pachytene spermatocytes. On the other hand, although few apoptotic germ cells were detected in biopsies from patients with obstructive azoospermia, increased levels of apoptotic germ cells were detected in spermatogenetic arrest. This increase in apoptotic germ cells was associated with increased levels of active caspase-3 in patients with spermatogenetic arrest, whereas the expression of XIAP and Smac/DIABLO was at similar levels in all groups. CONCLUSION(S) Active caspase-3 might be important in the apoptotic process observed in spermatogenetic arrest.

Sexualité et infertilité

Following the recent medical innovations, it is now possible to disassociate sexuality and reproduction. With contraception, people can have free sexuality without the fear of an unexpected pregnancy. Frequently, Assisted Reproductive Technologies (ART), with in vitro fertilization, can obtain a pregnancy without intercourse. There are three major problems concerning infertility and sexuality. Firstly, infertility because of a sexual disorder; secondly, sexual disorder induced by infertility diagnosis; thirdly, sexual disorder induced by ART. Praticians should be aware of possible existence of sexual problems to allow the couple to express them. Once diagnosed, these troubles can be treated by the pratician himself or the couple has to be referred to a psychologist or a sexologist.