Percutaneous cutting needle biopsies for histopathological assessment and sperm retrieval in men with azoospermia.

BACKGROUND: Twenty-three men (45 testes) with azoospermia underwent percutaneous testicular biopsy under local anaesthesia. METHODS: In all but one of the 45 testes two biopsies were taken close to each other, one with a 16 gauge (n = 44) and another with a 14 gauge (n = 45) cutting needle, both with a 19 mm notch. Three quarters of the tissue was used for histopathological assessment and one quarter for direct microscopy. RESULTS: The histopathological findings were similar between the two needles. The observations with direct microscopy corresponded with the histopathological assessments concerning the presence of mature spermatids in 41 of 45 (91%) biopsies using the 14 gauge and in 40 of 44 (91%) biopsies using the 16 gauge needle. There were no post-operative complications except for minimal pain and minor local swelling. CONCLUSIONS: Percutaneous material retrieved using 16 gauge and 14 gauge needles is sufficient for histopathological assessment, and the two needles are equally reliable for testicular sperm retrieval. However, needle biopsy with one puncture may not be representative of the entire testis.

Clinical outcome of treatment cycles using preimplantation genetic diagnosis for structural chromosomal abnormalities.

OBJECTIVES: To explore oocyte recovery, embryo quality, the number of transferable embryos and pregnancy rate after preimplantation genetic diagnosis (PGD) in patients with structural chromosomal aberrations. METHODS: PGD was performed in seven couples with Robertsonian translocations (Rob), eight couples with reciprocal translocations (Rec), two couples with inversions and one couple with a deletion. A total of 43 treatment cycles were carried out. RESULTS: A total of 14.2 oocytes per cycle were retrieved. Fertilisation and cleavage rates were 63% and 58%, respectively. Of the biopsied embryos 20% were transferable. Comparison of the Rob and Rec group revealed no significant differences in number of oocytes, fertilisation or cleavage rates. The number of transferable embryos after biopsy was significantly higher in the Rob group than in the Rec group. When embryo transfer (ET) was performed the pregnancy rate did not differ between the Rob and the Rec groups. Twenty-eight embryo transfers (one or two embryos) were carried out leading to eight clinical pregnancies (29% per ET): two twins, four singletons, one miscarriage and one ectopic pregnancy. All the children are carriers of balanced chromosomal aberrations. CONCLUSION: An acceptable pregnancy rate can be achieved among couples with structural chromosomal abnormalities.

Localization of oestrogen receptors alpha and beta in human testis.

Cellular localization of oestrogen receptor alpha (ERalpha) and beta (ERbeta) proteins were studied in human testis samples using immunohistochemistry, and the expression of the corresponding mRNA was examined with reverse transcription-polymerase chain reaction (RT-PCR). Seven men, aged 28-48 years, who underwent diagnostic testicular biopsy because of azoospermia or to give spermatozoa for intracytoplasmic injection for infertility treatment, donated tissue for the study. One of them had anejaculation but normally functioning testes, and one was diagnosed as having Sertoli cell-only syndrome (SCOS). In addition, expression of ERbeta protein was examined in one testis sample obtained from a man undergoing a sex change operation. Strong ERbeta immunoreactivity was detected in the nuclei of spermatogonia, spermatocytes and early developing spermatids. Elongating spermatids, mature spermatozoa, Sertoli and Leydig cells were all negative for ERbeta. The presence of ERbeta protein was confirmed in Western analysis. With RT-PCR, both wild-type ERbeta and ERbetacx, the isoform which represses wild-type ER function, were easily detected. In most cases, ERbetacx mRNA was more abundantly expressed than wild-type ERbeta. The patient with SCOS expressed neither ERbeta isoform. Neither ERalpha protein nor ERalpha mRNA was detected in any of the samples. We conclude that in the human testis, ERbeta is likely to be the ER that mediates the effects of oestrogen.

Highly abnormal cleavage divisions in preimplantation embryos from translocation carriers.

We have developed preimplantation genetic diagnosis (PGD) for carriers of chromosomal abnormalities using fluorescent in situ hybridisation (FISH). Here we present the detailed analysis of 64 biopsied, normally developing embryos obtained from four Robertsonian and three reciprocal translocation carriers in 11 treatment cycles of which four resulted in normal pregnancies (three simplex, one duplex). In order to investigate the degree of mosaicism and segregation mode in the embryos, the primary analysis of the biopsied cells was extended with the analysis of all cells from the non-transferred embryos. The analysis also included a second hybridisation with two additional probes, not involved in the translocation (chromosomes 1 and 9), in order to investigate the overall degree of mosaicism. Seventeen out of 64 analysed embryos were balanced for the chromosomes involved in the translocation and 14 of these were transferred. Forty-seven out of 64 embryos (73%) were mosaic regarding the chromosomes involved in the translocation and alternate segregation mode was the most common mode of segregation. Moreover, we have found a higher degree of mosaicism for the chromosomes involved in translocations as compared to control chromosomes. This difference was more pronounced for the embryos from reciprocal translocation carriers. The results, mechanisms, significance and implications of our findings are discussed.

Preimplantation genetic diagnosis of DiGeorge syndrome.

We report the first case of preimplantation genetic diagnosis used in order to avoid chromosomal imbalance in the progeny of a woman mildly affected by DiGeorge syndrome and carrier of a microdeletion of chromosome 22q11.2. In total, seven embryos were biopsied in three separate treatments and analysed by fluorescent in-situ hybridization (FISH). Of these, four were carrying the deletion, two were normal and in one the analysis was inconclusive. The diagnostic procedure was performed within 5 h. This allowed the biopsied embryos to be transferred the same day as the biopsy was taken (day 3). Two embryos were transferred in the third treatment, but no pregnancy was established. Patients with a 22q11 microdeletion, who have a 50% risk of transmitting the deletion to their offspring, can now be offered preimplantation genetic diagnosis using FISH for the detection of a 22q11 deletion.

Preimplantation genetic diagnosis of a large pericentric inversion of chromosome 5.

We report the first established pregnancy using preimplantation genetic diagnosis in order to avoid chromosomal imbalance in the progeny of a woman carrying a large inversion of chromosome 5. This is also the first time where it has been possible to study the distribution of balanced and unbalanced gametes in a female inversion carrier. In total, 23 embryos were biopsied in two separate treatments and analysed by fluorescent in-situ hybridization. Of these, 10 were unbalanced, nine were balanced and for four the analysis was inconclusive. The diagnostic procedure was performed within 3.5 h. This allowed the biopsied embryos to be transferred the same day as the biopsy was taken (day 3). Two embryos were transferred each time, and in the second treatment a twin pregnancy with two chromosomally balanced fetuses was established. Healthy twins were delivered at 34 weeks of gestation.

A comparison between open and percutaneous needle biopsies in men with azoospermia.

Open testicular biopsy is a classic method of investigation in men with azoospermia. Recently, percutaneous needle biopsy of the testis has been used in attempts to obtain material for histopathological diagnosis in such cases and to retrieve spermatozoa for intracytoplasmic sperm injection (ICSI). To determine whether a 19 gauge (G) and a 21G butterfly needle could be used for percutaneous aspiration of testicular tissue to determine the presence of mature spermatids and assess spermatogenesis, 10 patients (16 testes) and 12 patients (17 testes) underwent 19G or 21G needle biopsy respectively, immediately followed by open testicular biopsy, with both procedures under local anaesthesia. Biopsy with each needle size was compared with open biopsy. With the 19G needle, in the 14 cases where material was obtained there was full agreement with open biopsy regarding the presence or absence of mature spermatozoa, whereas with the 21G needle only nine of the 13 biopsies yielding material were predictive in this respect. Each needle size correlated poorly with open biopsy regarding evaluation of spermatogenesis. We conclude that percutaneous biopsy with a 19G butterfly needle is a quick and reliable method for demonstrating spermatozoa for ICSI. But for a detailed histopathological diagnosis, however, the needle biopsies gave poor results, whereas the material from the open testicular biopsies was assessable.

In-vitro fertilization and intracytoplasmic sperm injection in the treatment of infertility after testicular cancer.

Treatment of testicular cancer (TC) may cause infertility due to reduced sperm quality with or without an ejaculation problem. In cases of anejaculation or retrograde ejaculation, spermatozoa can be obtained by transrectal electroejaculation (TE) or testicular sperm extraction (TESE) and used for in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). In this study, 15 out of 17 couples evaluated for infertility after TC, underwent a total of 21 treatment cycles, resulting in 18 embryo transfers. Spermatozoa were obtained by TE in 16 cycles, by masturbation in three cycles and by TESE in one. In one cycle no spermatozoa were found using TESE. Fertilization and cleavage was achieved by IVF in seven cycles and ICSI in 11 cycles; average fertilization rates of 57 and 55% respectively were observed. Twelve clinical pregnancies occurred, of which 11 have been delivered or are ongoing. The ongoing pregnancy rate was 57% per cycle. These results show that infertility after testicular cancer can be treated effectively with IVF and that ICSI even permits treatment of patients who have severe oligozoospermia.

Application of single-needle blastomere biopsy in human preimplantation genetic diagnosis.

We have tested and subsequently successfully applied a single-needle approach to obtain blastomere biopsies from human preimplantation embryos for preimplantation genetic diagnosis (PGD). The method was first evaluated in a mouse system and shown to be compatible with a high degree of in vitro and in vivo development of biopsied mouse embryos. Furthermore, we showed that biopsied mouse embryos after transfer to recipient mice underwent implantation, normal development and delivery. Litters were followed through puberty and adulthood and shown to be normal with regard to sexual function and also a panel of biochemical and morphological parameters including organ histology. Successful human preimplantation diagnosis, followed by pregnancies and birth of healthy babies, was established with two out of three couples carrying a risk to transmit chromosomal abnormalities leading to severe disease. This is the first report of the successful use of a single-needle approach in human PGD. Considering its simplicity, we conclude that the single-needle approach is an attractive alternative for biopsies in PGD.

Epididymal and testicular sperm for intracytoplasmic sperm injection in the treatment of obstructive azoospermia.

BACKGROUND: The possibility of treating male infertility because of obstructive azoospermia has been poor, but intracytoplasmic sperm injection (ICSI) has given this type of infertility sufferer a new option. MATERIAL AND METHODS: In this study 13 couples with obstructive azoospermia were treated in a total of 19 stimulated IVF cycles. The men were between 27 and 45 (mean 33) years of age. Their partners, 24-39 (mean 31) years of age were treated according to routine IVF procedures, i.e. down regulation with buserelin followed by hyperstimulation with urofollitropin. Fertilization was obtained by ICSI. Two embryos were transferred on day two after the ovum pick up. Sperm were retrieved through microsurgical epididymal aspiration (MESA) in four cycles, percutaneous epididymal sperm aspiration (PESA) in three cycles and through testicular sperm extraction (TESE) in 12 cycles. RESULTS: The overall fertilization rate was 68%, with a cleavage rate of 82%.The fertilization rate was equal (68%) with epididymal and testicular sperm and the cleavage rate was 87%) and 80%, respectively. Embryos were obtained for embryo transfer (ET) in all cases and five pregnancies (one twin pregnancy) were established (26% per ET), three using epididymal sperm and two using testicular sperm. CONCLUSION: Infertility due to obstructive azoospermia can successfully be treated with epididymal sperm and ICSI. When epididymal sperm cannot be found sperm extracted from a testicular biopsy can be used. PESA and TESE are quicker and easier alternatives to MESA and can be performed on an outpatient basis with local anesthesia.

Transrectal electroejaculation in combination with in-vitro fertilization: an effective treatment of anejaculatory infertility after testicular cancer.

Treatment of non-seminomatous testicular cancer often leads to infertility due to anejaculation/retrograde ejaculation and poor sperm quality. In these men spermatozoa may be obtained by transrectal electroejaculation (TE), but the optimal strategy for assisted procreation in these couples is not known. Our aim was to examine whether TE and conventional in-vitro fertilization (IVF) would be successful. A total of 10 couples, with long-standing infertility due to anejaculation or retrograde ejaculation after treatment for testicular cancer 5-14 years earlier, were referred to our unit. All men underwent diagnostic TE under general anaesthesia. Spermatozoa were recovered in nine cases. The antegrade fraction was prepared and used for IVF. Sperm quality was variable and conventional IVF was considered impossible in three cases. Altogether six IVF treatment cycles in six couples resulted in five pregnancies, of which four resulted in a delivery and one resulted in a spontaneous abortion. One additional pregnancy is ongoing after transfer of cryopreserved embryos. The fertilization rate was 54% (33/61) and the cleavage rate was 97% (32/33). No complications relating to the procedure have been encountered.

Effects of insulin on free amino acids in plasma and the role of the amino acid metabolism in the etiology of diabetic microangiopathy.

To investigate if alterations of the amino acid metabolism may play a more important role in the etiology of diabetic microangiopathy than hitherto recognized, free amino acids in plasma were measured by means of high-performance liquid chromatography (HPLC) in healthy individuals (REF) and patients with insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). Isoleucine and leucine in IDDM were within normal limits, whereas they were significantly higher in NIDDM (P < 0.01 and P < 0.001, respectively). This was not due to age differences. In order to evaluate the impact of insulin on amino acid metabolism, amino acids were also measured in pregnant women (PREG) undergoing glucose tolerance tests as a screening for pregnancy diabetes and in patients with polycystic ovary syndrome (PCO) undergoing euglycemic insulin clamp tests. Insulin considerably reduced the amino acid concentration. Isoleucine and leucine were particularly depressed. On the whole there was strong covariance between the three branched-chain amino acids, isoleucine, leucine, and valine (P < 0.0001). There was no covariance between amino acid and glucose or HbA1c concentrations. A protein meal strongly stimulated insulin production (+55 mIU/liter), whereas a galactose meal revealed only a minor increase in insulin response (+12 mIU/liter) in contrast to a tolerance test with the same amount of glucose (+67 mIU/liter). It is concluded that disturbed amino acid metabolism may be a more important causative factor in the etiology of diabetic microangiopathy than hitherto recognized and, in addition, that this may affect the therapeutic approach in both IDDM and NIDDM patients.