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PubMed2002

Successful application of preimplantation genetic diagnosis for beta-thalassaemia and sickle cell anaemia in Italy.

BACKGROUND: In Italy, the autosomal recessive diseases beta-thalassaemia and sickle cell anaemia are so widespread that in some regions they can be defined as 'social diseases'. In this study, nine clinical applications of preimplantation genetic diagnosis (PGD) were performed for beta-thalassaemia and sickle cell anaemia on seven Sicilian couples and carriers of beta-globin gene mutations. METHODS AND RESULTS: The studied mutations were: Cd39, HbS, IVS1 nt1, IVS1 nt6 and IVS1 nt110. ICSI was performed with partner's sperm on 131 out of 147 retrieved oocytes, and this resulted in 72 zygotes; 32 embryos were successfully biopsied on day 3. The biopsied blastomeres were lysed and the beta-globin alleles amplified by nested PCR. The mutation diagnosis was performed by restriction enzyme digestion and reverse dot-blot. The amplification efficacy was 97.2%. The genotype study of non-transferred and surplus embryos showed that the allele drop-out rate was 8.6%. Seventeen embryos were transferred in utero on day 4. All couples received an embryo transfer; of the four pregnancies obtained, three resulted in live births and one miscarried at 11 weeks. Prenatal diagnosis at the 11th week and miscarriage material analysis confirmed the PGD results. CONCLUSIONS: These studies represent the first successful application of PGD for beta-thalassaemia and sickle cell anaemia in Italy.

Adult↗
PubMed2003

[Pregnancy and delivery after traumatic panhypopituitarism].

OBJECTIVE: We describe a case of 23 years old woman who, with a history of car accident at the age of 11 and severe head injury, attended our Center of Reproductive Medicine complaining of primary infertility. The accident preceded menarche and the development of inner genitalia (uterus 46 mm, cervix:corpus ratio 1:1) as well as secondary sexual development was not fully accomplished. Ovarian failure was accompanied by amenorrhea, if not substituted. Under replacement therapy regular menstruation was established. Basal hormonal levels was extremely low (FSH and LH 0.2-0.3 IU/l, PRL 0.5 ng/ml, estrogens 0.08 nmol/l, PRGE was not detected). With oral administration of micronized estradiol (Check et al., 2001) endometrial thickness of 9 mm was achieved. All of five retrieved oocytes were fertilized with partner sperm (normospermy) and three embryos were transferred. Already the first attempt was successful. Patient suffered with transitional anorexia and vomiting which could be handled conservatively. Hormonals level were normal. Patient substituted with Hydrocortison and Euthyrox. On January 2, 2003 the pregnancy was finished by caesarian section, one week before the delivery term (girl, 2970, Apgar score 10-10-10). DESIGN: Case report. SETTING: Center of Reproductive Medicine, Zlin. CONCLUSION: This care report demonstrates that recent progress in assisted reproduction field enables to manage successfully even exceptional pathological conditions.

Adult↗
PubMed1996

Delivery following intracytoplasmic injection of mature sperm cells recovered by testicular fine needle aspiration in a case of hypergonadotropic azoospermia due to maturation arrest.

This is the first reported delivery following intracytoplasmic sperm injection (ICSI) of mature live testicular sperm cells collected in a case of hypergonadotrophic azoospermia with maturation arrest. The 30 year old couple presented with primary infertility of 11 years duration, the man being submitted in childhood to five orchidopexy operations for the treatment of cryptorchism. He had elevated serum follicle stimulating hormone (FSH; 18.8 IU/I), an atrophic left testis and a normal sized right testis, the biopsy of which diagnosed maturation arrest and focal scarring. The couple refused donor insemination for religious reasons and the only option was an attempt at testicular sperm collection. Multiple testicular and epididymal fine needle aspirations were performed, using an aspiration handle loaded with 20 ml syringe and 21-23 gauge butterfly needles. The mature spermatozoa recovered were used to inseminate the oocytes by ICSI. Prior to this procedure, the patient's wife underwent ovulation induction using a long protocol of mid-luteal gonadotrophin-releasing hormone analogue/human menopausal gonadotrophin (GnRHa/HMG). At oocyte retrieval, ten oocytes were recovered. Eight live sperm cells were recovered from the aspirates of the right testis. Following ICSI into four metaphase II and two metaphase I oocytes, one mature oocyte was fertilized, cleaved and was transferred to the uterus 48 h after oocyte retrieval. The patient conceived and delivered a 3300 g boy at term. In conclusion, our results demonstrate that this novel approach should be considered in cases with hypergonadotrophic azoospermia due to testicular failure. Further experience is needed to establish the exact criteria for its use.

Adult↗
PubMed2004

Normal birth after single-embryo transfer in a patient with excessive polypronuclear zygote formation following in vitro fertilization and intracytoplasmic sperm injection.

OBJECTIVE: To report an unusual case of idiopathic zygotic polypronuclei. DESIGN: Case report. SETTING: Major urban infertility referral center. PATIENT(S): A 34-year-old woman with unexplained infertility. INTERVENTION(S): The patient underwent two cycles of controlled hyperstimulation and oocyte retrieval followed by in vitro insemination and intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Pregnancy and delivery of a normal infant following transfer of a single preembryo from an ICSI cycle in which only one zygote showed a normal pronuclear number (2PN) and 12 zygotes appeared polypronucleated (> or =3PN). RESULT(S): On the first IVF cycle, 23 oocytes were retrieved and inseminated with 240 x 10(3) motile sperm/mL, after which two zygotes showed a normal pronuclear number and 20 zygotes appeared polyploid with three to seven pronuclei. Transfer of two poor-quality day-3 preembryos following assisted hatching did not achieve pregnancy. On the subsequent ICSI cycle, 33 oocytes were retrieved, and 17 mature oocytes were subjected to ICSI, after which only one zygote showed 2PN and 12 zygotes appeared polyploid with three to eight pronuclei. The normally fertilized zygote developed into a poor-quality, day-3 embryo and was subjected to assisted hatching. Transfer of this preembryo resulted in an uneventful pregnancy and birth of a normal infant. CONCLUSION(S): Mechanisms other than polyspermia may result in polypronuclear development in some patients.

Adult↗
PubMedDate not supplied

Human sperm bioassay has potential in evaluating the quality of cumulus-oocyte complexes.

Human sperm bioassay is routinely used as a quality control check for the culture media. This is one of the three bioassays chosen by the College of American Pathologists (CAP) for interlaboratory proficiency testing to assess the standards of in vitro fertilization (IVF) and andrology laboratories. This study utilized sperm bioassay to assess the quality of cumulus-oocyte complexes (COCs) retrieved in IVF procedures COCs, harvested from the female partner of IVF couples, undergoing identical ovarian stimulation protocols, were individually inseminated with the sperm of the corresponding male partner. Sperm motility in sperm-COC cocultures were compared. Cocultures were established by inseminating the 103 COCs, retrieved from 18 IVF couples with 1 x 10(5) to 2 x 10(5) sperm of the corresponding male partners of the couples. In all 18 cases, the sperm were prepared identically using the Percoll wash method. The cocultures were maintained for 48 h but the oocytes were removed immediately after the fertilization check (approximately 16 h). The motility of sperm in the cocultures and in the insemination stocks were noted and 17 of 18 sperm stocks used for insemination had similar high preinsemination motility (90.2 +/- 5.0%). At 48 h the sperm motility had significantly decreased in the cocultures compared to the insemination stocks; 52.7 +/- 19.9% versus 67.2 +/- 10.4%. There was no difference in the motility among the small, medium, and large COCs (56.4 +/- 24.6%, 52.5 +/- 17.9%, and 50.8 +/- 20.9%, respectively). In 45% of IVF cases, the motility in cocultures varied widely, falling below as well as above that of their corresponding insemination stocks. Furthermore, the sperm motility varied among the cocultures in both pregnant and nonpregnant patients but the extent of variation appears to be greater in the latter. The inter-COC coculture sperm motility variation most likely is due to the differences in the quality of cumulus-oocyte complexes.

Adult↗
PubMed2001

Cryopreservation of human sperm.

Freezing of human sperm is considered a routine procedure in assisted reproductive technology (ART) laboratories. This article considers various aspects of cryopreservation of human sperm. Human sperm show a specific cryophysical behaviour and different sperm freezing protocols have been developed to avoid damage to the sperm cells. The damage can range from impaired motility and reduced viability to damage to the cellular organelles and effects at the molecular level, resulting in an impaired fertilizing potential. As testicular sperm are immature and only a small number can be retrieved, special techniques are required for successful freezing and thawing of these samples. Banking of human sperm has to be performed in a safe and controlled way and different guidelines are necessary to ensure that this is achieved.

Cell Survival↗
PubMed2003

Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection.

OBJECTIVE: To verify whether microinjection into retrieved oocytes of motile spermatozoa with morphologically normal nuclei, strictly defined by high power light microscopy (x >6000), improves the IVF/intracytoplasmic sperm injection (ICSI) pregnancy rate in couples with repeated ICSI failures. DESIGN: Comparative prospective study testing routine IVF/ICSI outcome parameters against those of modified ICSI based on morphological selection of spermatozoa with normal nuclei. SETTING: Male factor fertility laboratory and IVF center. PATIENT(S): Sixty-two couples, with at least two previous consequent pregnancy failed ICSI cycles, underwent a single ICSI trial preceded by morphological selection of spermatozoa with normal nuclei. Fifty of these couples were matched with couples who underwent a routine ICSI procedure at the same IVF center and exhibited the same number of previous ICSI failures. INTERVENTION(S): Standard ICSI and modified ICSI. MAIN OUTCOME MEASURE(S): ICSI pregnancy rate. RESULT(S): The matching study revealed that pregnancy rate after modified ICSI was significantly higher than that of the routine ICSI procedure (66.0% vs. 30.0%). CONCLUSION(S): Microinjection into retrieved oocytes of selected spermatozoa with strictly defined morphologically normal nuclei improves significantly the incidence of pregnancy in couples with previous ICSI failures.

Adult↗
PubMed1996

Polyploidy and failed fertilization in in-vitro fertilization are related to patient's age and gamete quality.

A review of 392 cycles of in-vitro fertilization (IVF) was carried out in order to identify whether any factors such as sperm concentration at insemination, sperm motility or morphology, oocyte grading, number of oocytes retrieved, patient age, follicular stimulation protocols or duration of follicular growth, could be associated with the incidence of polyploidy or completely failed fertilization. The majority of polypronuclear fertilizations occurred in mature oocytes and in patients < 37 years of age and the incidence of polyploidy was strongly associated with fertilization and pregnancy rates. Fertilization rates were highest with mature oocytes. A significant linear trend was observed with failed fertilization and sperm concentration, morphology and motility. High rates of failed fertilization were found in patients > 37 years of age and with one to five oocytes retrieved. No significant difference was seen in stimulation protocols and oocyte grading between oocytes that fertilized and those that did not. Cycles with good sperm morphology and motility, the presence of mature oocytes, the retrieval of a large number of oocytes and younger maternal age seem to provide the best chance for IVF success.

Adult↗
PubMed2004

Ejaculation failure on the day of oocyte retrieval for IVF: case report.

Unexpected ejaculation failure on the day of oocyte retrieval for IVF occurs once or twice a year in our Reproductive Medicine Unit, where approximately 500 oocyte retrievals are performed each year. Two clinical situations which occurred in 2001 are presented. In the first case, sperm were finally obtained by epididymal aspiration and resulted in the fertilization of five oocytes by ICSI. The transfer of two fresh embryos did not result in a pregnancy and the three supernumerary zygotes were cryopreserved. The male patient presented an anxio-depressive episode necessitating psychiatric hospitalization 1 week after the oocyte retrieval. In the second case, no sperm were obtained and the four oocytes were therefore lost. The couple went through a crisis in their relationship and tried another cycle of IVF 10 months later, after the preventive cryopreservation of a sperm sample. On the day of oocyte retrieval the patient was unable to produce a fresh sample but three zygotes were obtained through ICSI using the back-up cryopreserved sperm. Two embryos were transferred but no pregnancy ensued. The clinical decision-making processes for these two cases are described, as well as the measures employed to help prevent these unfortunate situations.

Adult↗
PubMed1998

Comparison of fertilization, cleavage and pregnancy rates of oocytes from large and small follicles.

Ovarian stimulation for in-vitro fertilization (IVF) causes development of several cohorts of follicles. At the time of oocyte collection, oocytes are thus retrieved from a wide range of follicles of different sizes and developmental stages. A relationship between size of follicles and pregnancy rates has earlier been demonstrated. The aim of the present study was to compare fertilization, cleavage and pregnancy rates between oocytes retrieved from large and small follicles in conventional IVF and intracytoplasmic sperm injection (ICSI). A total of 200 conventional IVF patients and 175 ICSI patients underwent oocyte retrieval where oocytes from both large and small follicles were collected. A follicle with a volume of > or = 2 ml, corresponding to a follicular diameter > or = 16 mm as determined by ultrasound, was regarded as a large follicle. Only one cycle from each patient was included. Fertilization and cleavage rates were calculated per patient for oocytes from large and small follicles. The mean fertilization and cleavage rates for conventional IVF and ICSI cycles were calculated. Comparison of pregnancy rates was performed for patients receiving embryos derived from oocytes of only large or only small follicles. For conventional IVF patients, fertilization rates were 71.4 and 58.1% (P < 0.01, Wilcoxon paired test) for oocytes of large and small follicles respectively. The corresponding cleavage rates were 95.4 and 93.9% respectively. The pregnancy rate for the two groups was 47% (60/127) and 15% (2/13) (P < 0.05, chi2 test). For ICSI patients the fertilization rate was 72.0 and 71.1% for oocytes of large and small follicles respectively. The corresponding cleavage rate was 93.0 and 91.1%. The pregnancy rate in the two groups was 41% (46/113) and 42% (5/12). The results show that oocytes from smaller follicles also yield fertilization and pregnancies, although in conventional IVF to a lesser extent than oocytes from larger follicles. For IVF cycles, a higher proportion of immature oocytes (which are normally not included in the ICSI procedure) in the group of oocytes from small follicles is most probably the explanation for the lower fertilization rate. The decrease in pregnancy rate with oocytes from small follicles in the IVF cycles was not observed in the ICSI cycles. The possibility of evaluating the degree of oocyte maturation prior to fertilization may be an advantage of the ICSI technique. This suggests that the disadvantages of oocytes from small follicles might be overcome by means of ICSI.

Adult↗
PubMed2003

High sex chromosome aneuploidy and diploidy rate of epididymal spermatozoa in obstructive azoospermic men.

PURPOSE: To evaluate the frequencies of sex chromosome aneuploidy and diploidy rate of epididymal spermatozoa from obstructive azoospermic men and its impact on intracytoplasmic sperm injection (ICSI) outcomes. METHODS: Epididymal spermatozoa retrieved from 24 obstructive azoospermic men and ejaculated spermatozoa from 24 fertile donors were analyzed using triple color fluorescence in situ hybridization (FISH) techniques, in order to investigate the rates of diploidy and aneuploidy for chromosomes 18, X and Y. RESULTS: Epididymal spermatozoa from obstructive azoospermic men had total sex aneuploidy, disomy 18, and diploidy rates significantly higher than ejaculated spermatozoa from normozoospermic fertile controls (1.44% vs 0.14%, 0.11% vs 0.02%, and 0.18% vs 0.02%, respectively; p < 0.005). There were no statistically significant differences in ICSI outcomes between the patients who had high and low epididymal sperm aneuploidy rate. CONCLUSIONS: Epididymal spermatozoa from obstructive azoospermic patients had an elevated sex chromosome aneuploidy and diploidy rate. The increased frequency of chromosomal abnormalities did not have a direct effect on the ICSI outcome.

Aneuploidy↗
PubMed2002

Predictive value of testicular histology in secretory azoospermic subgroups and clinical outcome after microinjection of fresh and frozen-thawed sperm and spermatids.

BACKGROUND: A retrospective study was carried out on 159 treatment cycles in 148 secretory azoospermic patients to determine whether histopathological secretory azoospermic subgroups were predictive for gamete retrieval, and to evaluate outcome of microinjection using fresh or frozen-thawed testicular sperm and spermatids. METHODS: Sperm and spermatids were recovered by open testicular biopsy and microinjected into oocytes. Fertilization and pregnancy rates were assessed. RESULTS: In hypoplasia, 97.7% of the 44 patients had late spermatids/sperm recovered. In maturation-arrest (MA; 47 patients), 31.9% had complete MA, and 68.1% incomplete MA due to a focus of early (36.2%) or late (31.9%) spermiogenesis. Gamete retrieval was achieved in 53.3, 41.2 and 93.3% of the cases respectively. In Sertoli cell-only syndrome (SCOS; 57 patients), 61.4% were complete SCOS, whereas incomplete SCOS cases showed one focus of MA (5.3%), or of early (29.8%) and late (3.5%) spermiogenesis. Only 29.8% of the patients had a successful gamete retrieval, 2.9% in complete and 77.3% in incomplete SCOS cases. In total, there were 87 ICSI, 39 elongated spermatid injection (ELSI) and 33 round spermatid injection (ROSI) treatment cycles, with mean values of fertilization rate of 71.4, 53.6 and 17%, and clinical pregnancy rates of 31.7, 26.3 and 0% respectively. CONCLUSIONS: Histopathological subgroups were positively correlated with successful gamete retrieval. No major outcome differences were observed between testicular sperm and elongated spermatids, either fresh or frozen-thawed. However, injection of intact round-spermatids showed very low rates of fertilization and no pregnancies.

Cell Size↗
PubMed2006

Comparison of embryo quality between sibling embryos originating from frozen or fresh oocytes.

Human embryo cryopreservation techniques allow storage of surplus embryos created during assisted reproduction procedures; however, the existence of these same surplus embryos has sparked further debate. What can be their fate once they are no longer desired by their parents, or if the parents are deceased? Thus, the level of interest in the cryopreservation of oocytes has increased, as has the necessity for further scientific study. This study had the objective of comparing embryo quality from 16 women who underwent intracytoplasmic sperm injection, where approximately half of the retrieved oocytes per cycle were inseminated fresh after collection, and the remainder cryopreserved for subsequent fertilization. Normal fertilization rates were not significantly different between the two oocyte-treatment groups. There was no significant difference in the frequency of good quality embryos (morphology grades I and II) on the second day of laboratory evaluation between embryos derived from the two oocyte-treatment groups. It is interesting to note that embryo transfer from fresh oocytes produced no pregnancies, which shows that even embryos derived from frozen oocytes that are fragmented or have a slower cleavage rate are viable embryos, capable of producing healthy babies.

Adult↗
PubMed1994

Does patient semen quality alter during an in vitro fertilization (IVF) program in a manner that is clinically significant when specific counseling is in operation?

PURPOSE: It has been shown that the stress of infertility may impair semen quality. Whether counseling can attenuate this affect is unproven. This study examines, in an IVF program setting, where specific counseling is in operation, whether semen collected on the day of oocyte recovery is significantly different from that obtained during the prior clinical suitability assessment of the couple. RESULTS: In the 125 consecutive couples examined, there were no significant overall differences in semen volume or sperm density. There was a significant increase in sperm motility on the day of oocyte retrieval (P < 0.001). Twenty-three patients (18.4%) showed an increase in quality, and 21 (16.8%) a decrease, on the day of oocyte recovery. The environment of production appeared to exert no influence. Fertilization failure occurred in seven couples, three (14%) of whom presented for the first time with decreased semen parameters on the day of IVF. This compares with 1 of 23 (4%) fertilization failure in those whose parameters increased. CONCLUSION: The semen quality of the vast majority of the patients studied in this highly counseled program does not appear to be significantly affected by the superstress of participation in the day of oocyte recovery.

Adult↗
PubMed2006

Histological evaluation of the human testis--approaches to optimizing the clinical value of the assessment: mini review.

Testicular biopsy is a crucial assessment in reproductive practice with diagnostic and prognostic importance for assisted reproductive technologies (ARTs) and risk of testicular neoplasia. Endocrine and genetic tests cannot reliably distinguish obstructive azoospermia (OA) from non-obstructive azoospermia (NOA) or predict recovery of mature spermatids by testicular sperm extraction (TESE). Currently, divergent histological reporting systems and the use of imprecise terminology seriously degrade the value of the literature on TESE recovery rates and hamper evaluation of treatments and research on genotype-phenotype relationships. The rising incidence of testis cancer and carcinoma in situ (CIS), especially in infertile populations, requires that every effort be made for its early detection. We provide a systematic approach to the histological classification of spermatogenic disorders and detection of CIS in adult patients. We evaluate a large consecutive series of bilateral biopsies from infertile men and report (i) the frequency of bilateral or discordant patterns that supports the use of bilateral biopsy for comprehensive evaluation and (ii) a high prevalence of mixed patterns, particularly within the hypospermatogenesis classification, that helps account for reported success of TESE. We propose a new diagnosis code for testicular biopsies that addresses the needs of ART clinicians and allows data storage and retrieval of value in clinical practice and research.

Azoospermia↗
PubMed2000

Incidence of congenital malformations in children born after ICSI.

The aim of this study was to determine the incidence of congenital malformations in a complete cohort of children born after intracytoplasmic sperm injection (ICSI). The medical records were retrieved for 1139 infants, 736 singletons, 200 sets of twins and one set of triplets. The total number of infants with an identified anomaly was 87 (7.6%), 40 of which were minor. The incidence of malformations in children born after ICSI was also compared with all births in Sweden using data from the Swedish Medical Birth Registry and the Registry of Congenital Malformations. For ICSI children, the odds ratio (OR) for having any major or minor malformation was 1.75 [95% confidence interval (CI) 1.19-2.58] after stratification for delivery hospital, year of birth and maternal age. If stratification for singletons/twins was also done, the OR was reduced to 1.19 (95% CI 0.79-1.81). The increased rate of congenital malformations is thus mainly a result of a high rate of multiple births. The only specific malformation which was found to occur in excess in children born after ICSI was hypospadias (relative risk 3.0, exact 95% CI 1. 09-6.50) which may be related to paternal subfertility.

Chromosome Aberrations↗
PubMedDate not supplied

Methylene blue but not indigo carmine is toxic to human luteal cells in vitro.

Methylene blue (MB) is reported to be teratogenic when injected intra-amniotically. Indigo carmine (IC) appears to be a safe alternative. To determine if MB has potential detrimental effects on ovarian tissue, we compared the effect of MB and IC on human granulosa luteal cell (GC) function in vitro. Human oocyte-cumulus complexes were obtained during in vitro fertilization cycles and one to three were placed in an organ culture dish. After insemination with sperm, oocytes were removed the day after retrieval and the attached GC were washed daily for 3 more days by changing 2 mL of culture medium. All the dishes were treated with human chorionic gonadotropin (hCG) for the next 24 h and progesterone (P) production during this interval was taken as baseline. Test chemicals were added with hCG for the next 48 h with daily media changes. The P production during the last 24 h of chemical treatment was expressed as a percentage of the baseline. MB significantly reduced P production whereas IC did not appear to have any effect. Moreover, under inverted microscopy more than 90% of the GC cells contained several small bluish intracellular granules when exposed to 0.01% MB but not 0.01% IC. These results indicate that MB may be taken up and processed by GC cells and inhibits P production. This finding adds to previous reports on the use of in vitro GC assay to identify potential reproductive toxicants. The clinical significance of this preliminary study needs further investigation.

Cells, Cultured↗