Jacqueline Selva

The single or dual administration of the gonadotropin-releasing hormone antagonist Cetrorelix* in an in vitro fertilization-embryo transfer program

OBJECTIVE To assess the ability of a GnRH antagonist (Cetrorelix, Asta Medica AG, Frankfurt, Germany) to prevent premature LH surges in an IVF-ET program using a simple protocol with one or two administrations. DESIGN Controlled ovarian hyperstimulation was carried out in 17 women with three ampules a day of hMG, starting on day 2 of the menstrual cycle. A dose of 5 mg of Cetrorelix was administered when plasma E2 levels were between 150 and 200 pg/mL (conversion factor to Sl unit, 3.671) per follicle of > or = 14 mm. A second injection was performed 48 hours later if the triggering of ovulation was not decided in the meantime. RESULTS Six patients received one injection and 11 patients received two administrations. Plasma LH levels showed a marked decrease and remained low after the administration of the GnRH antagonist. In six patients, the first administration of Cetrorelix was performed when a significant rise in LH plasma level was present. Even in these patients the GnRH antagonist was able to prevent an LH surge. The tolerance of the product was good. Six clinical pregnancies were obtained, of which four are ongoing (25% per ET). Two ongoing pregnancies were obtained after the transfer of a frozen-thawed embryo (35.3% per retrieval). CONCLUSIONS The GnRH antagonist Cetrorelix in a simple, unique or dual administration, protocol was able to prevent premature LH surge in all of the 17 patients studied. If these results are confirmed by larger, randomized studies, the good tolerance and efficacy that we observed suggest a bright future for this product is assisted reproductive technologies.

Correlation between DNA defect and sperm-head morphology

The utility of sperm DNA testing remains controversial. However, it may be helpful in couples with unexplained failures of multiple assisted reproductive techniques and/or recurrent abortions. This study analysed 10,400 spermatozoa of 26 patients for sperm-head morphology with high-magnification microscopy, DNA fragmentation and sperm chromatin decondensation. A significant negative correlation was demonstrated between sperm-parameters and abnormal sperm-head morphology as assessed by high magnification (score 0 according to this studys classification): concentration (r=-0.41; P=0.03), motility (r=-0.42; P=0.03), morphology (r=-0.63; P=0.0008). No correlation was found with DNA fragmentation. However, the sperm chromatin-decondensation rate of score-0 spermatozoa was twice as high as the controls (19.5% versus 10.1%; P<0.0001). This observation suggests that score-0 spermatozoa should not be selected for intracytoplasmic sperm injection.

A protocol for satisfying the ethical issues raised by oocyte donation: the free, anonymous, and fertile donors

A new protocol was developed to provide participants of our oocyte donation program with oocytes donated by donors who were not financially rewarded, were anonymous, and fertile. Each participant provided an oocyte donor selected among fertile friends or family members. The retrieved oocytes were anonymously exchanged between phenotypically matched donor-recipient pairs. In the first 30 months of activity, we obtained 111 embryos suitable for transfer or cryopreservation from 52 retrievals, and 40 embryo transfers (ETs) were performed. Recipients received oral Estradiol-valerate and vaginal micronized progesterone. Fifteen embryos were transferred fresh in 8 ETs conducted after donor-recipient synchronization. This resulted in four pregnancies, all ongoing (ongoing pregnancy rate 50% per transfer). Of the 96 cryopreserved embryos, 82 were thawed for ET, and 45 surviving embryos were transferred in 32 ETs. This resulted in eight pregnancies, with six ongoing or delivered (ongoing pregnancy rate 19% per transfer). The overall ongoing pregnancy rate of 25% per transfer indicates that our approach is a viable method for obtaining donated oocytes while respecting the ethical guidelines that recommend that donation of human gametes should be free, and from anonymous and fertile donors. Furthermore, guaranteeing anonymous oocyte donation had practical importance because, for many volunteer donors, it played a crucial role in their decision to donate.

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).

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.

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.

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.