Human immature dental pulp stem cells' contribution to developing mouse embryos: production of human/mouse preterm chimaeras.

OBJECTIVES: In this study, we aimed at determining whether human immature dental pulp stem cells (hIDPSC) would be able to contribute to different cell types in mouse blastocysts without damaging them. Also, we analysed whether these blastocysts would progress further into embryogenesis when implanted to the uterus of foster mice, and develop human/mouse chimaera with retention of hIDPSC derivates and their differentiation. MATERIALS AND METHODS: hIDPSC and mouse blastocysts were used in this study. Fluorescence staining of hIDPSC and injection into mouse blastocysts, was performed. Histology, immunohistochemistry, fluorescence in situ hybridization and confocal microscopy were carried out. RESULTS AND CONCLUSION: hIDPSC showed biological compatibility with the mouse host environment and could survive, proliferate and contribute to the inner cell mass as well as to the trophoblast cell layer after introduction into early mouse embryos (n = 28), which achieved the hatching stage following 24 and 48 h in culture. When transferred to foster mice (n = 5), these blastocysts with hIDPSC (n = 57) yielded embryos (n = 3) and foetuses (n = 6); demonstrating presence of human cells in various organs, such as brain, liver, intestine and hearts, of the human/mouse chimaeras. We verified whether hIDPSC would also be able to differentiate into specific cell types in the mouse environment. Contribution of hIDPSC in at least two types of tissues (muscles and epithelial), was confirmed. We showed that hIDPSC survived, proliferated and differentiated in mouse developing blastocysts and were capable of producing human/mouse chimaeras.

Microdissection testicular sperm extraction and IVF-ICSI outcome in nonobstructive azoospermia.

We evaluated the efficiency of microdissection testicular sperm extraction (MicroTESE) in patients with nonobstructive azoospermia (NOA) and their pregnancy outcomes in a programme based on in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI). Fifty-six MicroTESE procedures were performed in 53 patients with NOA. Pre-operative levels of luteinising hormone, follicle-stimulating hormone (FSH), testosterone and prolactin were obtained and a Doppler sonography examination was conducted. Sperm retrieval rate, mean age of female partner, mean ICSI and fertilisation rate, number and quality of embryos transferred, implantation, pregnancy and miscarriage rates were calculated. Samples for testicular histological analysis were taken trans-operatively in every case. Sperm retrieval rate, mean ICSI per case and fertilisation rate were 57.1%, 7.4% and 58.4% respectively. A significant difference in pre-operative testicular volume (P = 0.001), serum FSH (P = 0.008) and total testosterone levels (P = 0.021) was found in patients from whom sperm could be retrieved. Mean 1.9 type A embryos were transferred per cycle. Implantation, clinical pregnancy and miscarriage rates were 20%, 40% and 18.7% respectively. It is concluded that MicroTESE is a viable option for men with NOA, offering excellent results in couples undergoing IVF-ICSI. Pre-operative serum FSH, testicular volume and total testosterone levels may have a prognostic value, although more data are needed to determine their significance and whether or not patients should be excluded from an initial sperm retrieval attempt.

Laser-assisted ICSI: a novel approach to obtain higher oocyte survival and embryo quality rates.

BACKGROUND: Degeneration of oocytes occurs even when maximum care is exercised during ICSI, especially when the oolemma is very fragile and/or the zona pellucida is resistant. In order to be able to minimize the risk of degeneration associated with microinjection this study applied a new method: a microhole on the zona pellucida of the oocyte was drilled by laser beam just prior to ICSI to permit the penetration of the microneedle without any trauma. METHODS: A total of 32 patients (32 cycles) who had one or more previously failed ICSI cycles with a high degeneration rate of oocytes (>20%) were included in the study. Oocytes of the same patients were randomly divided into the study group [laser-assisted ICSI (LA-ICSI)] and the control group [conventional ICSI (C-ICSI)]. The outcomes of the cycles were compared and analysed. RESULTS: After LA-ICSI compared with C-ICSI, survival rates of oocytes were 99.6 and 84% (P < 0.0001), fertilization rates were 76.6 and 68.6% (not significant) and embryo development rates ( vertical line 6 cells on day 3) were 76.5 and 57.3% (P = 0.0024) respectively. CONCLUSIONS: Creating a microhole on the zona pellucida of the oocyte by laser beam prior to ICSI provides a less traumatic penetration of the injection needle into the ooplasm and results in lower degeneration and higher embryo development rates than C-ICSI in patients with fragile oocytes.

Rare association of ovarian implantation site for patients with heterotopic and with primary ectopic pregnancies after ICSI and blastocyst transfer.

Two cases of patients with ruptured ovarian pregnancies (P1 = ovarian heterotopic and P2 = primary ovarian ectopic) after intracytoplasmic sperm injection and blastocyst transfer are presented. Laparoscopy was performed on day 40 and day 27 after transfer in cases P1 and P2 respectively. In both cases the ectopic pregnancies were located on the left ovary and were successfully removed by laparoscopy preserving the ovaries. In case P1 the intrauterine pregnancy was not affected. A healthy boy was born after 37 weeks of pregnancy. In this way, potential fertility of the patients and the intrauterine pregnancy were maintained. These cases occurred during a series of blastocyst transfers in which 129 pregnancies were obtained. There were no cases of ovarian ectopic/heterotopic pregnancies from January 1996 to September 1999 in 814 pregnancies obtained from day 2 or day 3 embryo transfers. Because the ovarian ectopic pregnancies occurred in patients with day 5 embryo transfer who otherwise did not have any predisposing factors for ectopic pregnancy, it is advisable to conduct a large scale analysis of future data about the possible association between blastocyst-stage embryo transfer and the somewhat higher risk of unexpected complications of clinical outcome.

Monozygotic twins and transfer at the blastocyst stage after ICSI.

The incidence of monozygotic twinning (MZT) is higher in pregnancies conceived after assisted reproduction than after natural conception. Alterations, produced by ovarian stimulation, in-vitro culture conditions and specifically alterations of zona pellucida are mentioned as possible causes of this phenomenon. A retrospective review was performed of the incidence of MZT in pregnancies generated in our centre during the period of January 1996 to December 1999. This variable was compared in 129 gestations that resulted from blastocyst transfer (occurring from September 1998 to August 1999) with 814 pregnancies produced by transfers of 4- to 8-cell embryos. Follicular development was induced with human menopausal gonadotrophin and urinary FSH during 1996 and 1997 and with recombinant FSH during 1998 and 1999. Blastocysts were cultured in sequential media using S1 or G1 up to 72 h and S2 or G2 to day 5. Five of the 129 pregnancies generated by blastocyst transfers were complicated by MZT gestation (3.9%). In comparison, only six of 814 pregnancies occurred from 4- to 8-cell transfers (0.7%), a difference that is statistically significant (P< 0.001 with Yates correction). The results confirm an increase of MZT in pregnancies from intracytoplasmic sperm injection as compared to the natural incidence. Moreover, the frequency of MZT was significantly higher when transfers were performed at the blastocyst stage, suggesting that extended in-vitro culture of embryos may be associated with alterations of the zona pellucida and the hatching process.

Fertilizable oocytes reconstructed from patient's somatic cell nuclei and donor ooplasts.

The only assisted reproduction treatment now available for women with ovarian failure or irreparable oocyte defects is oocyte donation. However, some women experience psychological barriers to the recourse to donor oocytes, related to the lack of contribution of their proper genes to the progeny. A pilot study in humans suggests that this problem may be overcome by the development of techniques for haploidization of somatic cell nuclei, allowing the formation of new oocytes bearing the complete nuclear genome of the patient. Somatic cell nuclei were obtained from cumulus cells of a patient who failed to produce fertilizable oocytes and were transferred into enucleated oocytes (ooplasts) from a donor. Out of six ooplasts injected with the somatic cell nuclei and fertilized with spermatozoa from the patient's husband, signs of haploidization were detected in three oocytes, two of which subsequently started embryonic development and were cryopreserved for eventual future transfer to the genetic mother. These data show that human oocytes can be used for both reprogramming and haploidization of somatic cell nuclei, allowing reconstruction of genetically own oocytes for patients without, or with seriously disturbed, ovarian function.

Female age is an important parameter to predict treatment outcome in intracytoplasmic sperm injection.

OBJECTIVE: To assess the results obtained in our clinic with intracytoplasmic sperm injection (ICSI) in severe male factor infertility and failed fertilization and to determine if the age of the female has any impact on those results. DESIGN: Retrospective study. SETTING: Private high-complexity Human Reproduction Center. PATIENTS: One hundred five couples with a total of 114 procedures. Eighty-six were classified as severe male factor and 19 were classified as previous failed fertilization. INTERVENTIONS: Treatment was performed by ICSI. MAIN OUTCOME MEASURE: Normal fertilization, cleavage, and implantation, total, term, and ongoing pregnancy rates in the total population and in different age brackets. RESULTS: Excellent fertilization, cleavage, and implantation rates were obtained with this procedure (78%, 85%, and 13.5%, respectively). The total pregnancy rates were 43% and 46% per cycle and per transfer and 31.5% and 33.6% in terms of term and ongoing pregnancy rates. A significant reduction in implantation and total, and term and ongoing pregnancy rates was seen after the age of 35 years. CONCLUSIONS: The results obtained with ICSI are quite satisfactory with proper equipment and careful training. The age of the female is an important parameter in determining prognosis and treatment outcome.

Does treatment with testosterone undecanoate improve the in-vitro fertilizing capacity of spermatozoa in patients with idiopathic testicular failure? (results of a double blind study).

Seventy-seven couples in whom conventional in-vitro fertilization (IVF) had remained unsuccessful because of low fertilization rate and abnormal sperm characteristics were given either testosterone undecanoate 120 mg/day, or placebo during 3 months, after which a new IVF treatment was applied under identical technical conditions. There were no significant changes in sperm characteristics among the treated and placebo couples and the fertilization rate showed a similar increase after treatment in both groups. No significant difference in pregnancies occurred, with 32% pregnancies in the placebo controls and 17% among couples treated with testosterone undecanoate. It is concluded that testosterone undecanoate intake does not improve sperm characteristics, or the in-vitro fertilizing potential, or pregnancy rate over those observed in the placebo controls in cases with primary idiopathic testicular failure.

Tratamento com fertilizacao in vitro em retroejaculacao / Retroejaculation and treatment with in vitro fertilization - case report

Os autores descrevem o tratamento fertilizacao in vitro realizado com sucesso em um caso de infertilidade masculina por ejaculacao retrograda. Apos o preparo da bexiga urinaria, o meio HTF-Human TubalFluid foi injetado na bexiga, e a seguir solicitou-se ao paciente se masturbar e ter miccao natural em recipiente apropriado. A amostra coletada foi preparada, obtendo-se um semen com boa concentracao e motilidade. Com a estimulacao da ovulacao feita com o protocolo longo, utilizando GnRHa + hMG + hCG e aspiracao tranvaginal son controle ecografico obtiveram-se seis oocitos, dos quais quatro fertilizaram e foram transferidos ao utero. O suporte da fase lutea foi feito com progesterona oleosa e estradiol transdermico. No 14 dia pos-transferencia foi confirmada a gestacao pela dosagem de B-hCG e no 30 dia foi verificada a presenca de saco gestacional unico e topico com batimentos cardiacos presentes.

Pilot study with 120 mg Andriol treatment for couples with a low fertilization rate during in-vitro fertilization.

Treatment with 120 mg/day of Andriol (testosterone undecanoate; Organon, The Netherlands) was given to 11 men whose semen had either failed to fertilize, or had resulted in a less than 33% fertilization rate in a first in-vitro fertilization (IVF) trial. Repeat IVF at the end of a 3 month treatment period resulted in a highly significant increase in the number of oocytes fertilized from 4/95 (4.2%) before to 23/87 (26.4%, P less than 0.001) after Andriol treatment. One couple attained spontaneous conception during the second month of Andriol intake and three pregnancies occurred among the remaining 10 cases undergoing repeat IVF, for a total ongoing pregnancy rate of 36.4%. Sperm concentration but not motility increased in the native semen after Andriol intake, but there were no significant changes in sperm characteristics after preparation. The improvement in pregnancy rate contrasts favourably with the results obtained by repeat IVF of untreated historical controls. The result obtained in the present pilot study should encourage the performance of a larger, placebo-controlled investigation protocol.

Inseminaçäo intratubária por via vaginal (VITI): uma alternativa viável para reproduçäo assistida / Vaginal intratubal insemination (VITI): a viable alternative for assisted reproduction

Este estudo apresenta os resultados obtidos em nossa clínica com o uso de uma nova técnica de Reproduçäo Assistida, a inseminaçäo intra-tubária por via vaginal, sob controle ultrassonográfico (VITI). Estudaram-se os resultados obtidos com 82 pacientes que se consultaram em nossa Clínica e que apresentavam infertilidade devido a: a) Fator masculino (n=40): oligoastenospermia, teratospermia; b) Fator feminino (n=12): endometriose, distúrbios ovulatórios, obstruçäo tubária unilateral e sem outra causa aparente de infertilidade; c) Fatores masculino e feminino combinados (n=10); d) Esterilidade sem causa aparente (n = 20). A ovulaçäo foi induzida usando-se um protocolo combinado de citrato e gonadotrofina coriônica humana(hCG). Foram utilizados espermatozóides capacitados de amostras seminais frescas ou congelados (Banco de Semen). As pacientes foram monitorizadas e o diagnóstico de gravidez foi controlado pela dosagem de B-hCG e ultrassonografia. Em todas as 82 pacientes estudadas foi feita pelo menos uma tentativa de VITI, sendo que em algumas, de 2 a 6 tentativas. Um total de 13 gestaçöes clínicas foram conseguidas nessa série, com uma taxa geral de gestaçöes de 15,8%. Nenhuma das pacientes que se submeteram a somente uma tentativa (n=22) conseguiu engravidar no primeiro ciclo do tratamento. As taxas de gravidez para 2, 3 ou mais tentativas foram de 21,6% e 24,4%, respectivamente. Esses resultados indicam que a VITI pode ser aplicada com sucesso como técnica de reproduçäo assistida. Resta ser avaliado se esse método tem vantagens sobre outras técnicas, em particular sobre a IUI (Inseminaçäo Intrauterina)

Prognosis of varicocelectomy in the treatment of infertility, based on pre-surgery characteristics.

The semen characteristics, before and after varicocelectomy, were studied in 220 husbands from infertile couples, whose wives had no significant abnormalities. All couples were followed for one year or until the wife became pregnant. Sperm density, vitality, motility and percent with normal morphology were analysed before surgery. Based on these results, subjects were divided into 2 groups according to whether their semen had normal or abnormal characteristics. The group with semen had normal or abnormal characteristics. The group with semen parameters within normal limits showed significantly less improvement after surgery, and the posttreatment pregnancy rate was independent of the effect of surgery on the characteristics of sperm. Between 71 and 84% of the subjects with abnormal semen characteristics showed improvement after surgery, and between 56 and 72% of those who improved, impregnated their wives. Subjects whose abnormal semen characteristics did not improve after surgery, had a very low pregnancy rate.

[Treatment of male sterility (author's transl)]. / Tratamento da esterilidade masculina

In 330 cases of male sterility, the authors found in the seminal fluid 270 cases of quantitative alteration, 196 of qualitative alteration, 62 cases with biochemical disturbances and 122 patients with infections. After the specific treatment of these patients, the result was that 50 to 58.2% of them presented response for the quantitative, qualitative and biochemical alterations. In the infections, the index obtained was 85.2%. Among these 330 cases, they were obtained 73 cases of pregnancy (22.1%) with eight cases of abortion(10.9%).