Papaverine as a replacement for pentoxifylline to select thawed testicular or epididymal spermatozoa before ICSI.

OBJECTIVES: Pentoxifylline has been used to improve sperm motility in Assisted Reproductive Technology mainly by initiating sperm motility in immotile spermatozoa samples obtained surgically. Indeed, as Intracytoplasmic Sperm Injection leads to very poor results when using immotile gametes, pentoxifylline gives better results by easing the selection of viable sperm mobilized after incubation. In 2011, the French Haute Autorité de santé decided that pentoxifylline used for in vivo purpose proposed Insufficient Medical Service and pentoxifylline was thus withdrawn from the French materia medica. We here assessed the efficacy on spermatozoa motility and the safety of papaverine, another phosphodiesterase inhibitor, for the replacement of pentoxifylline. METHODS: Sixteen frozen-thawed epididymal or testicular samples displaying no or very poor spontaneous motility (≤5% total motility) were subjected to both pentoxifylline (3.6mM) and papaverine (93µM). A duplicate Mouse Embryo Assay and an In Vitro Fertilization Mouse Assay in duplo were used to discard any toxic effect of papaverine. RESULTS: Papaverine gave better results than pentoxifylline (mean total motility: 27% vs 23%, P<0.05). No Effect Level were observed in the two different Mouse Embryo Assays performed. CONCLUSION: Papaverine is a useful tool to replace pentoxifylline in ICSI programs to select viable spermatozoa in frozen-thawed sperm samples displaying no or very poor motility.

Relationship between even early cleavage and day 2 embryo score and assessment of their predictive value for pregnancy.

The aim of this study was, firstly, to define the different patterns of early cleavage (EC) observed at 26 h after either IVF or intracytoplasmic sperm injection (ICSI) and, secondly, to assess the predictive value of one of these patterns, even EC (EEC), on pregnancy rate in combination with day 2 embryo score. In the first part of the study, the relationship between three different EC patterns (EEC, uneven EC and EC with fragmentation of the day 2 embryo) and embryo morphology was determined. EEC was shown to be strongly associated with good embryo morphology. In the second part of the study, it was shown that EEC used in combination with embryo score improved selection of embryos for transfer. The presence of EEC significantly (P < 0.001) enhanced mean implantation rate in all transfer categories involving identically scored embryos, in both compulsory single embryo transfers and elective single embryo transfers. Multivariate analysis demonstrated that EEC and embryo score had strong complementary predictive value for pregnancy. Based on these findings, it was concluded that even though they are associated, EEC and embryo score could be combined to increase pregnancy rate, especially in elective single embryo transfer programmes.

Early cleavage: an additional predictor of high implantation rate following elective single embryo transfer.

The value of early cleavage (EC) assessment is still being debated. The aim of this prospective study was to examine the predictive value of EC assessment performed exactly 26 h after insemination by IVF or intracytoplasmic sperm injection (ICSI) in a programme of elective single embryo transfer (SET) performed at day 2. If day 2 scoring demonstrated several embryos with high implantation potential, an EC embryo was transferred preferentially. EC was assessed only during normal laboratory hours so that there were two groups: EC assessed, and EC not assessed, the latter being the control. A total of 277 elective SET were performed in women under 37 years undergoing their first IVF or ICSI cycle (mean age 30.5 years, range 21-37). The overall clinical and ongoing pregnancy rates were 40.1% (111/277) and 32.9% (91/277) respectively. Significantly higher overall clinical and ongoing pregnancy rates were obtained after transfer of an EC embryo than a non-EC embryo: 49.4 versus 33.3% (P < 0.05) and 42.4 versus 25.9% (P < 0.02) respectively. However there was no significant difference between the EC assessed and control groups: 40.4 versus 39.3% and 33.2 versus 32.1 respectively. These findings confirm the value of EC assessment for selection of embryos with high implantation potential.

[How to improve ART results? Is France behind other countries? Strategy for single embryo transfer: The value of embryo selection and embryo freezing]. / Comment améliorer nos résultats en AMP? La France est-elle en retard? Stratégie de transfert de l'embryon unique: la place du choix de l'embryon et de la congélation embryonnaire.

Multiple embryo transfer is associated with a high frequency of twin pregnancies with costly complications involving both mother and child. As a result high priority is currently being given to the development of single embryo transfer (SET) programs. France seems to be lagging behind Northern European countries in the development of SET and widespread use of SET will depend on convincing physicians that this policy will not have a negative impact on success rate, as has been the case for many protocols described in the literature as well as in our own experience. Our SET program includes patients less than 36 years of age undergoing their first FIV-ICSI. If two embryos showing satisfactory morphology are obtained, one is selected transferred and the other is systematically frozen. Selection for transfer is based on two criteria, i.e. observation of even early cleavage 26 hours after FIV-ICSI and evaluation of embryo morphology score on day 2. Embryo morphology score is based on the presence of four blastomeres and absence of blastomere irregularities and anucleated fragmentation. Last, a prerequisite for SET is an effective freezing program. A pregnancy rate of 13% per thawing was sufficient enough to obtain a cumulative pregnancy rate after SET (N = 205) and subsequent frozen embryo transfer (FET) similar to the cumulative pregnancy rate obtained after double embryo transfer (N = 394) and subsequent FET (46.3 vs 46.7%, NS). Twin delivery rate were respectively 2,6% after SET and 26,6% after double embryo transfer (P < 0.01).

[Elective single embryo transfer: a justified policy for selected patients]. / Transfert électif d'un seul embryon: une option justifiée pour une population de patientes sélectionnées.

OBJECTIVE: Prevention of twin pregnancies using elective Single Embryo Transfer (e-SET) is now considered by many Assisted Reproductive Techniques teams as a necessity. The aim of this study was to assess the efficacy of e-SET in a prospective manner in a selective population of patients using Take Home Baby Rate per couple as principal parameter. PATIENTS AND METHODS: This prospective study was conducted from January 2003 to December 2004. Elective Single Embryo was proposed to women above 37 years in their first IVF or ICSI attempt. It was then performed only in cases when at least one embryo with high implantation potential (score-4 embryo in our embryo scoring) was obtained for transfer and one more (score-3 or score-4 embryo) was available for freezing. RESULTS: e-SET was proposed and accepted in 225 couples (25% of eligible couples and 7.8% of total population) and was possible in 96 of these). Two embryos were transferred in all other eligible patients (Double Embryo Transfer group=DET). Cumulative delivery rate after fresh embryo transfers and, if necessary, after frozen-thawed embryo transfers were 39.5% per couple e-SET group and 41.7% in DET group (NS). On the other hand, the percentage of twin pregnancies was significantly different between the two groups (2.6% vs 26.6% respectively; P<0.01). DISCUSSION AND CONCLUSION: In women younger than 37 years in their first IVF/ICSI attempt, the elective transfer of only one embryo with high implantation potential strongly allowed to avoid twin pregnancies without any significant delivery rate decrease. This transfer policy is particularly efficient in laboratories displaying good results in their embryo freezing program.

Crude cumulative delivery rate following ICSI using intentionally frozen-thawed testicular spermatozoa in 51 men with non-obstructive azoospermia.

This prospective study evaluated the crude cumulative delivery rate following delayed intracytoplasmic sperm injection (ICSI) using spermatozoa recovered by testicular extraction (TESE) and intentionally frozen in men with non-obstructive azoospermia (NOA). This procedure can be termed 'cryoTESE-ICSI'. This study involved a series of 118 patients who underwent testicular biopsy for diagnosis of NOA in the period from January 1998 to December 2002. Testicular histology confirmed the diagnosis of NOA. Testicular parenchyma was obtained surgically from both testicles under general anaesthesia. Cryopreservation of spermatozoa was performed in 51 of 118 patients (43%). Ninety-nine delayed ICSI procedures were performed. Frozen-thawed suspensions were used in all cycles. Application of pentoxifylline was required to stimulate spermatozoa in 52% of cases. Fertilization, embryo transfer, and ongoing pregnancy rates were 60, 98 and 29% respectively. The crude cumulative delivery rate was 49% after two cycles and 57% after four cycles. A total of 39 healthy children were born in 29 deliveries. Thus, cryoTESE-ICSI is an effective procedure for routine use in patients with NOA. The main advantages of cryoTESE-ICSI are to (i) avoid repeated surgical biopsy, (ii) ensure the availability of spermatozoa when the ovarian stimulation cycle is begun, and (iii) allow programmed biopsy and therefore dissociate it from ICSI.

Outcome of ICSI in HIV-1-infected women.

BACKGROUND: Since 2001, French law has permitted the use of assisted reproductive technology in human immunodeficiency virus (HIV)-1 infected women under strict conditions. This report describes a preliminary series of seropositive women who underwent assisted reproduction treatment at our facility. To minimize contamination of culture media, equipment, and therefore of male gametes and embryos, we chose to perform ICSI in all cases. The outcome of ICSI was compared with the outcome in an age-matched group of non-HIV-1-infected women. Since several previous reports have indicated that HIV infection may be associated with a decrease in spontaneous fertility, our goal was also to assess the fertility status of the HIV-1-infected women entering our ICSI programme. METHODS: The French law governing the use of assisted reproduction protocols in HIV-1-infected women was strictly applied. The inclusion criteria were absence of ongoing disease, CD4((+)) count >200 cells/mm(3), and stable HIV-1 RNA level. Since mean age at the time of ICSI was higher in HIV-1-infected women than in the overall group of non-HIV-infected women, we compared outcome data in HIV-1-infected women (group I) to a group of non-HIV-1-infected women matched with regard to age and follicle retrieval period (group II) as well as to the overall group of women who underwent ICSI at our institution (group III). RESULTS: A total of 66 ovarian stimulations was performed in 29 HIV-1-infected-infected women. The percentage of cancelled cycles was higher in infected women than in matched controls (15.2 versus 4.9%, P < 0.05). The duration of ovarian stimulation (13.3 versus 11.7 days, P < 0.05) and amount of recombinant FSH injected (2898 versus 2429 IU, P < 0.001) were also higher in infected women. The number of retrieved oocytes, mature oocytes, and embryos obtained as well as embryo quality was similar in all groups. The fertilization rate was higher in infected women than in matched controls (67 versus 60%, P < 0.01). The pregnancy rate was not significantly different between groups I and II (16.1 versus 19.6%) in spite of the fact that the number of embryos transferred was purposefully restricted in the HIV-1-infected group to minimize multiple pregnancy (2.0 versus 2.4, not significant). CONCLUSION: The results of this preliminary series of ICSI cycles in HIV-1-infected women indicate that optimal ovarian stimulation is slightly more difficult to achieve than in matched seronegative women. However, when criteria for oocyte retrieval were fulfilled, ICSI results were similar to those of age-matched controls.

Sequence family variant loss from the AZFc interval of the human Y chromosome, but not gene copy loss, is strongly associated with male infertility.

BACKGROUND: Complete deletion of the complete AZFc interval of the Y chromosome is the most common known genetic cause of human male infertility. Two partial AZFc deletions (gr/gr and b1/b3) that remove some copies of all AZFc genes have recently been identified in infertile and fertile populations, and an association study indicates that the resulting gene dose reduction represents a risk factor for spermatogenic failure. METHODS: To determine the incidence of various partial AZFc deletions and their effect on fertility, we combined quantitative and qualitative analyses of the AZFc interval at the DAZ and CDY1 loci in 300 infertile men and 399 control men. RESULTS: We detected 34 partial AZFc deletions (32 gr/gr deletions), arising from at least 19 independent deletion events, and found gr/gr deletion in 6% of infertile and 3.5% of control men (p>0.05). Our data provide evidence for two large AZFc inversion polymorphisms, and for relative hot and cold spots of unequal crossing over within the blocks of homology that mediate gr/gr deletion. Using SFVs (sequence family variants), we discriminate DAZ1/2, DAZ3/4, CDY1a (proximal), and CDY1b (distal) and define four types of DAZ-CDY1 gr/gr deletion. CONCLUSIONS: The only deletion type to show an association with infertility was DAZ3/4-CDY1a (p = 0.042), suggesting that most gr/gr deletions are neutral variants. We see a stronger association, however, between loss of the CDY1a SFV and infertility (p = 0.002). Thus, loss of this SFV through deletion or gene conversion could be a major risk factor for male infertility.

Depot GnRH agonist versus the single dose GnRH antagonist regimen (cetrorelix, 3 mg) in patients undergoing assisted reproduction treatment.

The objective of this study was to compare, in a centre with previous experience of gonadotrophin-releasing hormone (GnRH) antagonist use, single administration of a GnRH antagonist [cetrorelix (Cetrotide) 3 mg] with a single administration of a GnRH agonist [Decapeptyl Retard 3.75 mg] in patients undergoing assisted reproduction treatment (n = 307 and 364 respectively). GnRH agonist was administered on the first day of menses, while cetrorelix was administered when the largest follicle reached 14 mm. Ovarian stimulation was performed with recombinant human FSH (r-hFSH; 150-225 IU/day). Human chorionic gonadotrophin (HCG, 10,000 IU) was administered when at least two follicles reached a mean diameter > or =18 mm. Over 90% of patients in both groups reached the criteria for HCG administration and underwent oocyte retrieval and embryo transfer. Duration of FSH therapy (9.95 versus 11.25 days) and cumulative dose of r-hFSH (1604 versus 1980 IU) were significantly reduced (P < 0.01) in the cetrorelix 3 mg group. The number of oocytes retrieved was lower (8.5 versus 11.2; P < 0.01) with cetrorelix, but the number of embryos replaced was similar (2.2 versus 2.3; NS). The pregnancy rates per oocyte retrieval were the same, 24.5%, in the antagonist and agonist groups. This study indicates that although fewer oocytes are recovered, similar pregnancy rates can be achieved with a GnRH antagonist compared with a GnRH agonist. Additionally, a single dose of 3 mg cetrorelix was administered in 84% of patients, thus being simpler and more convenient for patients. Cetrorelix 3 mg may thus be proposed as a first choice for preventing both a premature LH surge and detrimental rises in LH during ovarian stimulation prior to assisted reproduction treatment.

Embryo score is a better predictor of pregnancy than the number of transferred embryos or female age.

OBJECTIVE: To compare the value of 17 IVF variables, including a new mean score of transferred embryos (MSTE), in predicting pregnancy rate. DESIGN: Retrospective study. SETTING: Private IVF unit. PATIENT(S): Women who underwent 10,000 embryo transfers. MAIN OUTCOME MEASURE(S): Duration of infertility, type of infertility, female age, rank of IVF attempt, type of ovarian treatment, progesterone level, sperm count, sperm motility, sperm morphology, number of retrieved oocytes, number of mature oocytes, maturation rate, number of embryos obtained, fertilization rate, number of transferred embryos, cumulative embryo score (CES), mean score of transferred embryos (MSTE), and pregnancy rate. RESULT(S): Outcome of IVF-ET was significantly correlated with female age, type of infertility, number of retrieved oocytes, number of mature oocytes, maturation rate, embryos obtained, fertilization rate, transferred embryos, CES, and MSTE. Multivariate analysis demonstrated that MSTE was a better predictor of pregnancy than the number of transferred embryos and female age. CONCLUSION(S): Embryo quality is the best predictor of pregnancy. The embryo score described herein should be used in IVF-ET programs to choose the best embryos for transfer.

The human Y chromosome genes BPY2, CDY1 and DAZ are not essential for sustained fertility.

Deletions of the AZFc interval of the human Y chromosome are found in >5% of male patients with idiopathic infertility and are associated with a severely reduced sperm count. The most common deletion type is large (>1 Mb) and removes members of the Y-borne testis-specific gene families of BPY2, CDY1, DAZ, PRY, RBMY2 and TTY2, which are candidate AZF genes. Four exceptional individuals who have transmitted a large AZFc deletion naturally to their infertile sons have, however, been described. In three cases, transmission was to an only son, but in the fourth case a Y chromosome, shown to be deleted for all copies of DAZ, was transmitted from a father to his four infertile sons. Here we present a second family of this latter type and demonstrate that an AZFc-deleted Y chromosome lacking not only DAZ, but also BPY2 and CDY1, has been transmitted from a father to his three infertile sons. Polymerase chain reaction (PCR) and Southern blot analyses revealed no difference in the size of the AZFc deletion in the father and his sons. We propose that the father carries rare alleles of autosomal or X-linked loci which suppress the infertility that is frequently associated with the absence of AZFc.

Pentoxifylline initiates motility in spontaneously immotile epididymal and testicular spermatozoa and allows normal fertilization, pregnancy, and birth after intracytoplasmic sperm injection.

PURPOSE: Pentoxifylline (PF) has been used to enhance sperm motility in many in vitro fertilization programs. The twofold purpose of this study was to determine whether PF stimulates fresh or frozen epididymal and testicular totally immotile spermatozoa and whether it can be used to select viable spermatozoa for intracytoplasmic sperm injection (ICSI). METHODS: To test the effect of PF on motility, 10 samples of totally immotile spermatozoa were incubated for 10 min with 3.6 mM PF. Motility was initiated in all cases (14.8% mean motility after PF treatment of five samples of fresh or frozen epididymal spermatozoa and 13.6% mean motility of five samples of fresh or frozen testicular spermatozoa). To assess PF for selection of viable spermatozoa before ICSI, we compared the outcome of ICSI in 20 cycles using fresh or frozen epididymal or testicular PF-treated immotile spermatozoa and 139 control ICSI using fresh or frozen epididymal or testicular spontaneously motile spermatozoa. RESULTS: Fertilization rates were similar in the PF and control groups (45.2% vs. 51.0%). Embryo quality and division stages at the time of transfer were comparable. Six pregnancies occurred in PF-ICSI group (30.0% per cycle vs. 26.6% in control group) including two deliveries of healthy children and four ongoing pregnancies.

The critical region of overlap defining the AZFa male infertility interval of proximal Yq contains three transcribed sequences.

The position of deletion breakpoints in a series of four AZFa male infertility patients has been refined using new markers derived from BAC clone DNA sequence covering the AZFa male infertility interval. The proximal half of the AZFa interval is occupied by pseudogene sequences with homology to Xp22. The distal half contains an anonymous expressed sequence tag (named AZFaT1) found transcribed in brain, testis, and skeletal muscle and the DFFRY and DBY genes. All the patients have AZFaT1 and DFFRY deleted in their entirety and three patients additionally have DBY deleted. The three patients with AZFaT1, DFFRY, and DBY deleted show a severe Sertoli cell only syndrome type I phenotype, whereas the patient that has retained DBY shows a milder oligozoospermic phenotype. The expression of DBY in a cell line from this latter patient is unaltered; this shows that it is the loss of genes lying within the deletion that is responsible for the observed oligozoospermia. RT-PCR analysis of mouse testis RNA from normal and XXSxr(a) mice (devoid of germ cells) has shown that Dby is expressed primarily in somatic cells and that the level of expression is unaltered during germ cell differentiation. This contrasts with Dffry where no transcripts are detectable in XXSxr(a) mouse testis and expression occurs specifically in testis mRNA in a germ cell dependent fashion.

Teratozoospermia influences fertilization rate in vitro but not embryo quality.

The purpose of this study was to retrospectively compare the overall results and embryo quality in 102 cycles of in-vitro fertilization (IVF)-embryo transfer using normal frozen donor semen (group D) and 94 cycles of IVF-embryo transfer using husbands' teratozoospermic sperm (group T). Donor semen was purchased from men with proven fertility and normal semen parameters. Teratozoospermia was defined in group T as the presence of <20% of normal spermatozoa in semen on the day of oocyte retrieval. Exclusion criteria were a sperm count <10 x 10(6)/ml or with <10% progressive motility. Fertilization rate, transfer rate and number of transferred embryos per cycle were significantly lower in the teratozoospermic group (45 vs 72%, 66 vs 96%, 1.7 vs 2.9%, respectively). Pregnancy rate per cycle was also lower, but not significantly (18 vs 28%). However, pregnancy rate per transfer, implantation rate per transferred embryo and take home baby rate were comparable (27 vs 30%, 15 vs 15%, 21 vs 24%, respectively). Similarly, embryo quality in terms of number of embryos displaying fragmentations or irregular cells, cleavage stages and embryo scores were comparable. When group T was divided into two subgroups according to sperm count (group T1: sperm count = 10-20 x 10(6)/ml; group T2: sperm count >20 x 10(6)/ml), there was no difference between them with regard to fertilization rate, pregnancy rate or embryo quality. This study confirms low pregnancy rate per cycle in IVF-embryo transfer using teratozoospermic semen, but demonstrates for the first time that embryo quality and viability are not impaired. It is proposed that the poor pregnancy rate per cycle obtained is due only to the poor fertilization rate, and to the subsequent limited choice of embryos to be transferred.

[Value of an embryo score to predict implantation]. / Intérêt d'un score embryonnaire pour le pronostic implantatoire.

The prognostic value of the score of embryo transfer (SET) was assessed on 1079 embryo transfers in a homogene population of patients. The SET is obtained adding the embryo score (ES) of each transferred embryos. ES is based both on the conventional parameters of embryo morphology, and on the 4-cell stage which is the optimum division stage regarding implantation. The SET is strongly correlated with the pregnancy rate (p < 10-5), and is, with the mean score of transferred embryos (MSTE), the most sensitive and specific parameter of all studied parameters. However, embryo quality seems not to be correlated with miscarriages.

Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers.

The purpose of this study was to devise an embryo score to predict the likelihood of successful implantation after in-vitro fertilization (IVF). Unlike most studies dealing with the influence of embryo stage and morphology on pregnancy, our study was based on single rather than multiple embryo transfers. A total of 957 single embryo transfers were carried out. No delivery was obtained after any of the 99 transfers using 1-cell embryos or embryos obtained after delayed fertilization. In the remaining 858 transfers, the embryos had cleaved. Higher pregnancy rates were obtained with embryos displaying no irregular cells (11.7 versus 6.9%; P < 0.01) and embryos displaying no fragmentation (11.5 versus 8.1%; P < 0.05). The 4-cell embryos implanted 2-fold more often than embryos with more or less cells (15.6 versus 7.4%; P < 0.01). Based on these observations, we devised a 4-point embryo score in which embryos are assigned 1 point each if they (i) are cleaved, (ii) present no fragmentation, (iii) display no irregularities, and (iv) have four cells. Both pregnancy rate and take home baby rate were significantly correlated with embryo score. Each point of this score corresponds to a 4% increase in pregnancy rate. Interestingly, pregnancy rate was significantly lower in women aged > 38 years (8.2 versus 11.4%; P < 0.05), even though embryo quality was similar regardless of age. Single embryo transfer allowed us to define a simple and useful embryo score to choose the best embryo for transfer to optimize IVF and embryo transfer outcome. The use of this embryo score could decrease multiple pregnancies after multiple embryo transfers.

[Intracytoplasmic injection and embryo quality: comparison with conventional in vitro fertilization]. / Injection intracytoplasmique et qualité embryonnaire: comparaison avec la FIV conventionnelle.

We report here the results of a study comparing the quality of the embryos obtained after conventional IVF and after ICSI (respectively 872 and 459 embryos for 184 and 108 cycles). In the ICSI group, the female age was lower than in the IVF group, the oestradiol level on the day of hCG injection was higher, so that the number of retrieved oocytes and the number of mature oocytes. The policy of transfer being the same in the two groups, the mean number of transferred embryos was identical. The percentage of embryos without anucleate fragments, the percentage of embryos without irregularities, the percentage of 1, 2, 3, 4 or 5-cell embryos and the distribution of embryos in the 5 embryo scores were similar. In both IVF and ICSI groups, the transfer score (sum of the embryo scores of each transferred embryo) was higher for the patients who achieved pregnancy.

[Comparison of optical analysis and computerized analysis of sperm movement in a program of 316 in vitro fertilizations]. / Comparaison de l'analyse optique et de l'analyse automatisée du mouvement des spermatozoïdes (AAMS) dans un programme de 316 fécondations in vitro.

The purpose of this study was to compare visual and computerized assisted semen analysis (CASA) in an in vitro fertilization program. Both methods were performed on the day of 316 oocyte retrievals. CASA was performed using a Hamilton 7.2 device adjusted in accordance with the settings defined by a group of 14 French centers. Despite a few discrepancies, the correlation between visual and CASA sperm counts was significant (p < 0.001), so that between the two total motility assessments. The same correlation (Pearson's test) was observed between the fertilization rate and the parameters obtained by visual analysis and CASA. VAP, VSL, VCL and ALH exhibited threshold values under which the mean fertilization rate was below 50%. This study demonstrates the satisfactory reliability of CASA although it cannot yet replace visual analysis.