Marianne Bergere

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.

Soluble HLA-G in IVF/ICSI embryo culture supernatants does not always predict implantation success: a multicentre study

Several reports have described an association between the presence of soluble human leukocyte antigen G (sHLA-G) in human embryo culture supernatants (ES) and implantation success. However, not all studies agree with these findings. To further document this debate, a multicentre blinded study was performed to investigate, on a large number of IVF ES and ICSI ES, whether sHLA-G is a useful criterion for embryo selection before transfer. A total of 1405 ES from 355 patients were collected from three assisted reproductive technique (ART) centres and evaluated for their sHLA-G content in a single laboratory, using a chemiluminescence enzyme-linked immunosorbent assay. In only one centre was a significant association between sHLA-G-positive ES and successful implantation established (P = 0.0379), whereas no such association was observed in the other centres. It was found that the percentages and concentrations of sHLA-G-positive ES varied between centres, depending on culture media and ART conditions. The percentage of sHLA-G-positive ES was significantly higher in IVF ES than ICSI ES (P < 0.001 and P < 0.01 for two centres). These data demonstrate that substantial variations of sHLA-G content in ES occur between different ART centres, highlighting the influence of several technical parameters that differ from one centre to another.

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.

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).

Cytogenetic analysis of uncleaved oocytes after intracytoplasmic sperm injection

PurposeThis work analyzes the causes of cleavage failure after intracytoplasmic sperm injection (ICSI) and the effect of the procedure on the chromosomes of the oocytes.MethodsNinety-seven uncleaved oocytes from 39 patients with severe male infertility or repeated IVF failure were fixed; 79 were analyzable. We checked the decondensation stage of spermatozoa nucleus and the chromosomal abnormalities of the oocytes.ResultsAmong the fixed oocytes, the spermatozoa nucleus was present in 97% of the cases, and it was undecondensed in 89% of the cases, showing no evolution at all. A low rate (2.6%) of premature chromosome condensation (PCC) of the spermatozoa and a low rate (2.5%) of female diploïdy were observed. Among the oocytes that could be karyotyped, we observed a high rate (45%) of chromosome breakage.ConclusionICSI fertilization failure was due mostly to the complete lack of evolution of the spermatozoa nucleus. Oocyte selection before ICSI seemed to lower the PCC rate. The high rate of oocyte chromosomal breakage rate has to be confirmed.

Cryopreserved Immature Mouse Oocytes: A Chromosomal and Spindle Study

AbstractPurpose: Cryopreservation of human oocytes might provide an alternative approach to freezing supernumerary embryos obtained during IVF. This process, performed on immature denuded prophase I mouse oocytes, was investigated. Methods: We first investigated the capacity of frozen, immature, murine oocytes to continue in vitro maturation after thawing. We then evaluated the risk to offspring from chromosomal damage by cytogenetical and cytological (spindle) analysis. Finally, we attempted to determine the reasons for and the stage of maturation failure. Results: A total of 700 immature oocytes was frozen, 629 (90%) were recovered intact after thawing, and 53% extruded the first polar body, versus 74% for the control group. Freezing was not accompanied by an increase in aneuploidy in maturing oocytes (18 and 15% for thawed and control oocytes, respectively). Consequently, the first meiotic division occurred normally, without an increase in nondisjunction. Spindle analysis demonstrated only a few abnormalities (15 and 2% for thawed and control oocytes, respectively) incompatible with further development. Oocytes arrested during in vitro maturation were mainly at the metaphase I stage (64 and 76% for thawed and control oocytes, respectively). Whereas 17% of thawed oocytes were blocked before the formation of the first meiotic spindle, this never occurred in the control group. Conclusions: Immature murine oocytes can withstand cryopreservation, which is encouraging for future human application of this technique.

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.