Analysis of the evolution of chromosome abnormalities in human embryos from Day 3 to 5 using CGH and FISH.

The use of interphase fluorescent in situ hybridization (FISH) has shown that a large number of human embryos exhibit chromosomal abnormalities in vitro. The most common abnormality is mosaicism which is seen in up to 50% of preimplantation embryos at all stages of development. In this study, comparative genomic hybridization (CGH) was used to analyse 1-2 cells biopsied on Day 3 of development while the rest of the embryo was cultured until Day 5. Embryos were spread on Day 5 and analysed by FISH using probe combinations that varied depending on the CGH result, to investigate the progress of any abnormalities detected on Day 3. A total of 37 frozen-thawed embryos were analysed in this study. One gave no CGH or FISH results and was excluded from analysis. Six embryos failed to give any FISH result as they were degenerating on Day 5. Thirty embryos provided results from both techniques. According to the CGH results, the embryos were divided into two groups; Group 1 had a normal CGH result (13 embryos) and Group 2 an abnormal CGH result (17 embryos). For Group 1, three embryos showed normal CGH and FISH results, while 10 embryos were mosaic after FISH analysis, with various levels of abnormalities. For Group 2, FISH showed that all embryos were mosaic or completely chaotic. The combination of CGH and FISH enabled the thorough investigation of the evolution of mosaicism and of the mechanisms by which it is generated. The main two mechanisms identified were whole or partial chromosome loss and gain. These were observed in embryos examined on both Day 3 and 5.

A comparison between quarter, partial and total laser assisted hatching in selected infertility patients.

BACKGROUND: The object of this study was to evaluate the efficacy of laser assisted hatching (LAH) of embryos on implantation and pregnancy rates of a selected group of infertility patients. METHODS: A total of 322 cycles using LAH was undertaken in our Centre between June 1998 and September 1999. Patients were offered LAH if they fell in either one or more of the following categories: (i) Patients over 37 years of age undergoing either IVF or intracytoplasmic sperm injection (ICSI) treatment cycles; (ii) patients with more than 2 previous treatment cycle failures; (iii) patients undergoing frozen embryo replacement cycles and (iv) women who were considered to be poor responders. The initial results of totally breaching the zona pellucida (total LAH; group 1) did not meet with our expectations. We subsequently modified the technique to thinning one area of the zona pellucida (partial LAH; group 2) and this thinned area was then extended to a quarter segment (quarter LAH; group 3). RESULTS: In group 1, the pregnancy rate was 14.6% with a clinical pregnancy rate of 5.2%. In group 2 the pregnancy rate was 20.9% with a clinical pregnancy rate of 18% and for patients in group 3 the pregnancy rate was 29.0% with a clinical pregnancy rate of 22.1%. CONCLUSIONS: Overall there was firm statistical evidence that the pregnancy and clinical pregnancy rates arising from quarter LAH were higher in comparison with partial and total LAH.

Gestational surrogacy: a feasible option for patients with Rokitansky syndrome.

Rokitansky syndrome is a developmental defect characterized by agenesis of the uterus and vagina but normal gonads and secondary sexual characters. It is not commonly transmitted as a dominant genetic trait. Surrogacy, which is legally and ethically accepted in the UK and other countries, has made it possible for the patients with this syndrome to have their own genetic children. Six patients with Rokitansky syndrome underwent 11 ovarian stimulation cycles that resulted in 11 fresh and three frozen embryo transfer procedures into six prospective surrogate mothers. Both commissioning and surrogate couples were properly screened and counselled and their treatment was approved by the clinic internal review committee (ethics committee). The treatment cycles resulted in six clinical pregnancies (42.9% pregnancy rate per embryo transfer and 54.5% per oocyte retrieval) and three live births (21. 4% per embryo transfer, 27.3% per retrieval and 50% per patient). Gestational surrogacy is a viable treatment for patients with Rokitansky syndrome. Such patients should be well informed and supported to be able to have a family using their own genetic gametes.

Studies of percutaneous epididymal sperm aspiration (PESA) and intracytoplasmic sperm injection.

Four distinct studies were carried out using two data sets of percutaneous epididymal sperm aspiration (PESA) and intracytoplasmic sperm injection (ICSI) procedures performed from March 1993 to January 1997. In study A, an analysis of 181 ICSI treatment cycles following PESA revealed a successful epididymal sperm retrieval rate of 83%. It confirmed that PESA is an effective sperm retrieval method and the associated ICSI pregnancy rate (35% per embryo transfer) compared favourably with that of other sperm retrieval methods. In study B, the relevance of a prior diagnostic PESA procedure was ascertained by comparing the sperm retrieval rates in two groups of patients having their first ICSI treatment cycle with spermatozoa retrieved through PESA. Group B1 (n = 50) had diagnostic PESA prior to the ICSI treatment cycle PESA procedure, unlike patients in group B2 (n = 64) who did not. The sperm retrieval rate in the treatment cycle procedure was not different at 90 and 82.8% for groups B1 and B2 respectively. However, the discontinuation of diagnostic PESA is fraught with problems including liability to medico-legal sanctions. In study C, analysis of 177 treatment cycles involving PESA and ICSI revealed a successful sperm retrieval rate by PESA of 82% in the first cycle, 93% in the second, 96% in the third and 100% in the fourth cycle. The same trend was evident when sperm retrieval was examined in relation to each of the epididymides. Retrieved spermatozoa were found to be motile in 67-100% of cases and the frequency of samples containing motile spermatozoa did not decrease with increase in the number of PESA attempts. These results show that PESA does not jeopardize future epididymal sperm retrieval. In study D, the outcome of treatment with ICSI using ejaculated spermatozoa (305 cycles) (group D1) was compared with that of ICSI using spermatozoa obtained through PESA (54 cycles) (group D2). The median age of women in the two groups of couples was similar (34 years). In group D1, 70% of metaphase II oocytes were fertilized compared with 61% in group D2 (P < 0.01). The cleavage rate and the median numbers of transferred and cryopreserved embryos were similar in both groups. There was no significant difference between the clinical pregnancy rates (33 and 42% in groups D1 and D2 respectively). Our results show that the outcome of PESA-ICSI treatment compares favourably with that of ICSI using ejaculated spermatozoa.

The efficacy of local anaesthesia for percutaneous epididymal sperm aspiration and testicular sperm aspiration.

A total of 37 percutaneous epididymal sperm aspiration (PESA) and/or testicular sperm aspiration (TESA) procedures were performed under local anaesthesia (LA) on 34 men between June and November 1996. Local anaesthesia was achieved by injecting 10 ml of 1% lignocaine solution along the sides of the vas deferens near the external inguinal ring (spermatic cord block). Sperm retrieval was successful in 92% of the procedures. Of the 37 procedures, in 29 the patients felt either no pain or mild discomfort while in six they experienced moderate but tolerable pain. Analgesia was incomplete in two procedures and was supplemented with i.v. sedation. Vasovagal reflex in two procedures was reversed by i.v. atropine. In 24 procedures patients felt relaxed, whilst in 13 they felt anxious. In 32 procedures the patients expressed overall satisfaction. If the procedure was to be repeated, after 29 procedures the patients requested LA again, while after four procedures they preferred i.v. sedation and after four were undecided. LA is adequate for PESA and TESA in a large proportion of patients. Prior discussion of LA technique with the patient is necessary. Back-up facilities for i.v. sedation and atropine should be available.

Utilization of retrieved oocytes as an index of the efficiency of superovulation strategies for in-vitro fertilization treatment.

An analysis of 581 in-vitro fertilization treatment cycles was carried out to determine the pattern of utilization of retrieved oocytes. Patients were divided into five groups depending on the number of retrieved oocytes. The mean fertilization rate of 57% was broadly similar amongst the groups but the proportion of retrieved oocytes that produced embryos of a quality suitable for transfer or cryopreservation (the cycle efficiency index) fell significantly with increase in the retrieved oocyte number. However, the pregnancy rate increased with the number of retrieved oocytes (13-38%). It is important to determine the point at which advantages of multifollicular development are outweighed by the potential for complications. Increased utilization of retrieved oocytes will decrease the need for production of a large number of oocytes.

Utility of percutaneous epididymal sperm aspiration in situations of unexpected obstructive azoospermia.

Surgical sperm retrieval through percutaneous epididymal aspiration was used to manage effectively unexpected obstructive azoospermia on the day of oocyte retrieval.

Embryo developmental stage at transfer influences outcome of treatment with intracytoplasmic sperm injection.

PURPOSE: Our purpose was to determine if embryo cell stage at the time of intrauterine transfer correlates with pregnancy rate in patients treated with intracytoplasmic sperm injection (ICSI). METHODS: We conducted a retrospective analysis of 455 embryo transfer cycles following ICSI and 304 conventional in vitro fertilization (IVF) and embryo transfer cycles in women aged 40 years or less. Abstracted information included grading of the embryo cell stage and quality at the time of transfer. RESULTS: The overall ICSI pregnancy rate was 30.8%, while that of conventional IVF was 29.3%. However, the ICSI pregnancy rate fell to 9.3% for embryo transfers taking place at the two-cell stage but increased to 35.8% when at least one embryo had more than two cells, and this difference was statistically significant (P < or = 0.0001). The pregnancy rate following conventional IVF was 22.0% when only two-cell embryos were transferred and 32.0% when at least one of the embryos had more than two cells, but this difference in pregnancy rates was not significant (P > 0.05). CONCLUSIONS: The stage of embryo development at transfer appears to exert a powerful influence on the successful establishment of pregnancy after ICSI.

Experience with gestational surrogacy as a treatment for sterility resulting from hysterectomy.

A retrospective study was carried out to assess the potential of 16 hysterectomized women to achieve surrogate pregnancies. A total of 11 patients completed 16 cycles of assisted conception treatment incorporating in-vitro fertilization and gestational surrogacy. Three other women failed to respond to ovulation induction while two more patients produced few oocytes which also failed to fertilize. Six host mothers became pregnant thereby giving a pregnancy rate of 37.5% (6/16) per patient and embryo transfer and 27.3% (6/22) per cycle of treatment commenced. Two women later miscarried, three have given birth to twins and the remaining host has delivered a male infant. The commissioning mother's age was closely related to occurrence and continuation of pregnancy in the host. Hysterectomized women demonstrate varying patterns of response to assisted conception treatment but gestational surrogacy generally appears to be a feasible option especially in younger patients.

Evidence from a salvaged treatment cycle supports an aetiology for the empty follicle syndrome that is related to terminal follicular developmental events.

Oocyte retrieval in a stimulated in-vitro fertilization treatment cycle was unsuccessful when inadvertently carried out 12 h after the administration of human chorionic gonadotrophin (HCG) injection. Repeat follicular aspiration at 36 h post-HCG injection recovered 20 oocytes, out of which 16 metaphase-II eggs were subjected to intracytoplasmic sperm injection and eight became fertilized. Uterine transfer of three cleaving embryos resulted in a singleton pregnancy which went to term and a healthy female infant was delivered. Our experience shows that in addition to issues of HCG bioavailability to the developing follicles, the temporal relationship between HCG administration (or the luteinizing hormone surge) and follicular aspiration is also an important consideration in the determination of the aetiology of the empty follicle syndrome.

Results of percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in two major groups of patients with obstructive azoospermia.

Percutaneous epididymal sperm aspiration (PESA) and intracytoplasmic sperm injection (ICSI) were carried out in patients with congenital bilateral absence of the vas deferens (CBAVD) and men with failed reversal of vasectomy (FRV). PESA was successful in 55 out of 62 patients with CBAVD (89%) and in 57 out of 60 men with FRV (95%). The fertilization rates after ICSI (53 and 55%), cleavage rates (70 and 76%) and pregnancy rates (36 and 32%) did not differ significantly between the two respective groups (CBAVD and FRV). PESA and ICSI are effective both in patients with CBAVD and in those with FRV.

Cumulative experience of percutaneous epididymal sperm aspiration (PESA) with intracytoplasmic sperm injection.

OBJECTIVE: Our objective was to evaluate the recovery rate of spermatozoa from the epididymis using a percutaneous aspiration technique and to assess the fertilisation rate after intracytoplasmic sperm injection. MATERIALS AND METHODS: Fifty-four patients with azoospermia had a total of 59 cycles at IVF with intracytoplasmic sperm injection (ICSI). The cause of the azoospermia was failed vasectomy reversal in 23 cases, congenital absence of the vas in 22 cases, partial testicular failure in 5 cases, and retrograde ejaculation in 2 cases, while the remaining 2 patients had erectile disorders. RESULTS: A total of 741 oocytes was collected and 521 metaphase II oocytes were subsequently microinjected. Normal fertilisation occurred in 274 oocytes (52.6%), and of these, 234 cleaved (85.4%). In 54 cycles, embryo transfer of more than one embryo occurred (91.5%) and a total of 155 embryos was replaced. The pregnancy rate was 30.5% per cycle and 33.3% per embryo transfer. The implantation rate was 14.2%; failure of fertilisation occurred in two cycles, while in three other cycles the embryos did not cleave. CONCLUSIONS: Percutaneous epididymal sperm aspiration can be used successfully to retrieve sperm in men with azoospermia due to obstructive, or nonobstructive, disorders. The technique is simple, cost-effective, and associated with fewer complications than an open microsurgical operation.

Simplified sperm retrieval and intracytoplasmic sperm injection in patients with azoospermia.

OBJECTIVE: To evaluate the rate of recovery of spermatozoa from the epididymis using a percutaneous aspiration technique and to assess the fertilization rate following intracytoplasmic sperm injection (ICSI). PATIENTS AND METHODS: Forty-two patients with azoospermia underwent a total of 46 treatment cycles of in vitro fertilization (IVF) and ICSI. The sperm used for ICSI was retrieved percutaneously by fine-needle aspiration and syringe suction (percutaneous epididymal sperm aspiration, PESA) from the epididymis in 28 patients (mean age 34.9 years), over 32 cycles. Six patients underwent microsurgical sperm aspiration (MESA) and in the remaining eight patients, neither percutaneous aspiration nor MESA yielded suitable sperm and spermatozoa extracted from testicular biopsy were used. RESULTS: A total of 362 oocytes were collected and of those, 286 (79%) were subjected to ICSI. Of the injected oocytes, 49 (17.2%) were damaged, 138 (48.3%) achieved normal fertilization and, of those, 112 (81.2%) cleaved. A total of 67 embryos were transferred and 18 more were suitable for cryopreservation. Of the 25 cycles with embryo transfer, eight resulted in a pregnancy and of these, one miscarried. The pregnancy rate was 25% per cycle and 32% per embryo transfer. The implantation rate was 12%. CONCLUSIONS: This extensive series of PESA/ICSI cycles indicates that PESA can be used successfully to retrieve spermatozoa in patients with azoospermia. The technique is simple, cost-effective and is associated with fewer complications compared to an open microsurgical procedure.

Factors influencing the outcome of in-vitro fertilization with percutaneous aspirated epididymal spermatozoa and intracytoplasmic sperm injection in azoospermic men.

In-vitro fertilization (IVF) by intracytoplasmic sperm injection (ICSI) with spermatozoa retrieved by percutaneous epididymal sperm aspiration (PESA) is a novel, simple and effective treatment for azoospermic men. In all, 38 azoospermic men had an IVF/PESA/ICSI cycle. A total of 42 cycles were performed. The aetiology of azoospermia was classified as failed vasectomy reversal (12 patients/16 cycles), inflammatory obstruction (five patients/five cycles), partial testicular failure (five patients/five cycles) and bilateral congenital absence of vas (16 patients/16 cycles). Adequate sperm preparations for ICSI were obtained from 38 of the 42 treatment cycles (90%). The mean fertilization rate was 32.7%, and fertilization occurred in 35 cycles (92.0%). Embryo transfer was performed in 13 out of 14 cycles (93%) in men with a failed vasectomy reversal, four out of five cycles in men with an inflammatory obstruction (80%), four out of four cycles in men with a partial testicular failure (100%), and 14 out of 15 cycles in men with a bilateral congenital absence of vas (93%). The overall pregnancy rate per two or three embryos transferred was 28.6 and 26.3% per treatment cycle respectively. The sperm parameters of the final pooled sperm aspirate preparations varied widely among the four aetiological groups. These parameters were of no value in predicting the fertilization or pregnancy rates (P > 0.05), and neither was the embryo cleavage rate.

The value of single versus repeated insemination in intra-uterine donor insemination cycles.

Pregnancy rates per cycle of intra-uterine donor insemination following ovulation induction were compared retrospectively for those patients having a single, and those having repeated insemination using frozen donor semen. Single insemination was performed in 69 cycles in which 15 women became pregnant (pregnancy rate = 22%). Of 65 cycles in which repeated insemination was performed, 16 women became pregnant (pregnancy rate = 25%). This difference in pregnancy rates was not statistically significant (chi 2 = 3.6, P = 0.84). We conclude that cycle fecundity may not be increased by repeating insemination.