Marc Bailly

Genetic polymorphisms influence the ovarian response to rFSH stimulation in patients undergoing in vitro fertilization programs with ICSI.

Introduction Obtaining an adequate number of high-quality oocytes is a major challenge in controlled ovarian hyperstimulation (COH). To date, a range of hormonal and clinical parameters have been used to optimize COH but none have significant predictive value. This variability could be due to the genetic predispositions of single-nucleotide polymorphisms (SNPs). Here, we assessed the individual and combined impacts of thirteen SNPs that reportedly influence the outcome of in vitro fertilisation (IVF) on the ovarian response to rFSH stimulation for patients undergoing intracytoplasmic sperm injection program (ICSI). Results Univariate analysis revealed that only FSHR, ESR2 and p53 SNPs influenced the number of mature oocytes. The association was statistically significant for FSHR (p=0.0047) and ESR2 (0.0017) in the overall study population and for FSHR (p=0.0009) and p53 (p=0.0048) in subgroup that was more homogeneous in terms of clinical variables. After Bonferroni correction and a multivariate analysis, only the differences for FSHR and ESR2 polymorphisms were still statistically significant. In a multilocus analysis, only the FSHR and AMH SNP combination significantly influenced oocyte numbers in both population (p<0.01). Discussion We confirmed the impact of FSHR and ESR2 polymorphisms on the IVF outcome. Furthermore, we showed for the first time that a p53 polymorphism (which is already known to impact embryo implantation) could influence the ovarian response. However, given that this result lost its statistical significance after multivariate analysis, more data are needed to draw firm conclusions. Only the FSHR and AMH polymorphism combination appears to influence mature oocyte numbers but this finding also needs to be confirmed. Materials and Methods A 13 gene polymorphisms: FSHR(Asn680Ser), p53(Arg72Pro), AMH(Ile49Ser), ESR2(+1730G>A), ESR1(−397T>C), BMP15(−9C>G), MTHFR1(677C>T), MTHFR2(1298A>C), HLA-G(−725C>G), VEGF(+405G>C), TNFα(−308A>G), AMHR(−482 A>G), PAI-1 (4 G/5 G), multiplex PCR assay was designed to genotype women undergoing ICSI program. We analyzed the overall study population (n=427) and a subgroup with homogeneous characteristics (n=112).

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.

Effects of low concentrations of inhibin B on the outcomes of testicular sperm extraction and intracytoplasmic sperm injection

OBJECTIVE To study the effects of undetectable inhibin B concentrations on the outcomes of testicular sperm extraction (TESE) and of intracytoplasmic sperm injection (ICSI). DESIGN Retrospective study. SETTING Obstetrics, gynecology, and reproductive biology departments. PATIENT(S) We carried out TESE on 75 men with nonobstructive azoospermia: 42 men had an inhibin B concentration of or = 15 pg/mL (group 2). Twenty-five ICSI cycles were carried out using sperm from men in group 1 (group A1), and 35 ISCI cycles were carried out using sperm from men in group 2 (group A2). The outcomes of ICSI in groups A1 and A2 were compared with those of 81 ICSI cycles performed for obstructive azoospermia (group B). INTERVENTION(S) Testicular sperm extraction, testicular spermatozoa cryopreservation, and ICSI. MAIN OUTCOME MEASURE(S) Testicular sperm extraction outcome, pregnancy, and delivery. RESULT(S) Sperm were significantly less likely to be successfully recovered from men in group 1 than from those in group 2 (21% vs. 48%). The inhibin B concentration was significantly lower in men in whom TESE failed, but the FSH concentration did not differ. The implantation rate per embryo transferred was twofold lower in group A1 (7.4%) than in group B (16%), but this difference is not statistically significant. CONCLUSION(S) Patients with undetectable inhibin B concentration should be informed of the low chances of positive testicular biopsy, and more embryos should be transferred to improve the success rate.

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.

High-magnification selection of spermatozoa prior to oocyte injection: confirmed and potential indications

Intracytoplasmic morphologically selected sperm injection (IMSI) involves the use of differential interference contrast microscopy at high magnification (at least ·6300) to improve the observation of live human spermatozoa (particularly by showing sperm head vacuoles that are not necessarily seen at lower magnifications) prior to intracytoplasmic sperm injection (ICSI) into the oocyte. However, a decade after IMSI’s introduction, the technique’s indications and ability to increase pregnancy and/or birth rates (relative to conventional ICSI) are subject to debate. In an attempt to clarify this debate, this work performed a systematic literature review according to the PRISMA guidelines. The PubMed database was searched from 2001 onwards with the terms ‘IMSI’, ‘MSOME’ and ‘high-magnification, sperm’. Out of 168 search results, 22 relevant studies reporting IMSI outcomes in terms of blastocyst, pregnancy, delivery and/or birth rates were selected and reviewed. The studies’ methodologies and results are described and discussed herein. In view of the scarcity of head-to-head IMSI versus ICSI studies, the only confirmed indication for IMSI is recurrent implantation failure following ICSI. All other potential indications of IMSI require further investigation.

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.

Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities linked to chromatin condensation failure

Since an embryos ability to grow to the blastocyst stage and implant can be improved by selection of a normal spermatozoon with a vacuole-free head, this study set out to determine the nature of small sperm vacuoles observed under high magnification (>×6300). For 15 infertile men with various sperm profiles, high-magnification microscopy was used to select motile, morphometrically normal spermatozoa with no vacuoles (n=450) or more than two small vacuoles (each of which occupied less than 4% of the heads area; n=450). Spermatozoa acrosome reaction status and degree of chromatin condensation were analysed. Three-dimensional deconvolution microscopy was used to accurately image the nucleus and acrosome at all depths in all spermatozoa. In all 450 spermatozoa with small vacuoles, the latter were seen to be abnormal, DNA-free nuclear concavities. Spermatozoa with small vacuoles were significantly more likely than vacuole-free spermatozoa to have noncondensed chromatin (39.8% versus 9.3%, respectively; P<0.0001). There was no significant difference between the two groups of spermatozoa in terms of acrosome reaction status. No association between chromatin condensation and acrosome reaction status was observed. Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities related to failure of chromatin condensation.

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.

Successful pregnancy and delivery after testicular sperm extraction despite an undetectable concentration of serum inhibin B in a patient with nonobstructive azoospermia

OBJECTIVE To describe a successful pregnancy and delivery after testicular sperm extraction (TESE) despite an undetectable concentration of serum inhibin B in a man with nonobstructive azoospermia. DESIGN Case report. SETTING Obstetrics and gynecology and reproductive biology departments. PATIENT(S) A 31-year-old woman and a 32-year-old man with nonobstructive azoospermia and an undetectable inhibin B serum level. INTERVENTION(S) TESE, testicular spermatozoa cryopreservation, intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S) Pregnancy and delivery. RESULT(S) Successful pregnancy and delivery of a normal healthy child following a third ICSI cycle with frozen-thawed spermatozoa extracted from the testis. CONCLUSION(S) This case report shows that there is no minimal level of inhibin B below which TESE is always unsuccessful. The delivery of a normal healthy baby is strong evidence to perform TESE in these circumstances.