Pain experience during transvaginal aspiration of immature oocytes.

BACKGROUND: We wanted to determine the pain conception prior to, during, and after transvaginal recovery of immature oocytes for in vitro maturation (IVM). MATERIAL AND METHODS: Fifty women received 1 g Paracetamol and 0.125 mg Halcion as preanalgesics. During the oocyte pick-up para-cervical blockage (PCB) with 5 ml Citanest x2 was given, in addition to 1.5-2 ml Raphiphene intravenously. The pain was measured by using a visual analog scale (VAS). The expected pain was recorded just before the oocyte pick-up. Right after the oocyte pick-up the actual pain was measured and again 1/2 hour, and 1-11/2 hour later. Furthermore, the pain was recorded every second hour after discharge and every third hour the next day. RESULTS: Forty-three women experienced less pain than expected (p=0.003). The pain conception was correlated to the number of punctures through the vagina (p=0.012). Fifteen patients received analgesics postoperatively, and all patients were discharged less than 2 hours after the procedure without complaints. None noted any discomfort or pain after 30 hours following the oocyte pick-up. CONCLUSION: Although the IVM oocyte pick-up procedure involved puncture of very small follicles that required numerous punctures through the vagina, the experienced pain was significantly less than the expected pain, and the procedure was well accepted.

Incidence of apoptosis in granulosa cells from immature human follicles.

The aim of this study was to investigate the incidence of apoptosis in granulosa cells from immature human follicles undergoing in vitro maturation (IVM) and to compare the incidence of apoptotic granulosa cells (i) between FSH-primed and unprimed normal ovaries and (ii) between polycystic and normal ovaries. Furthermore, the incidence of apoptosis was related to maturation and subsequent fertilization and cleavage of the oocytes from the corresponding ovary. Seventy women undergoing 70 IVM cycles were included. Group 1 consisted of patients with normal ovaries (n = 52) and group 2 consisted of patients with polycystic ovaries (n = 18). Patients in group 1 were subdivided into two groups according to priming with FSH before aspiration. In group 1a (n = 27 cycles) oocytes were obtained in unstimulated cycles. In group 1b (n = 25 cycles) oocytes were obtained after priming with recombinant FSH for 3 days initiated on day 3 after spontaneous menstruation. In group 2 all patients were primed with recombinant FSH for 3 days before aspiration. Aspiration was performed transvaginally and cumulus-enclosed oocytes were matured for 28-30 h before fertilization. Granulosa cells were collected from follicular aspirates. An APOPTAG detection kit was used to stain the granulosa cells and to detect apoptosis. The incidence of apoptosis in granulosa cells was decreased in follicles from FSH-primed normal ovaries compared with follicles from unprimed normal ovaries and FSH-primed polycystic ovaries. No difference was found between granulosa cells from FSH-primed polycystic ovaries and granulosa cells from unstimulated normal ovaries. No differences in maturation rate, fertilization rate, cleavage rate and implantation rate were observed when oocytes from a polycystic ovary were compared with oocytes from an unstimulated normal ovary. In unstimulated cycles, the ovaries were grouped according to the presence of a dominant follicle. The incidence of apoptosis was significantly higher in granulosa cells from an ovary without a dominant follicle compared with granulosa cells from an ovary with a dominant follicle. The rates of maturation, fertilization and cleavage did not differ between the two groups.

Maternal serum supplementation in culture medium benefits maturation of immature human oocytes.

This study compared the rates of maturation, fertilization, cleavage and pregnancy among oocytes matured in medium containing either human serum albumin (HSA) or maternal serum. Immature oocytes were obtained from 51 consecutive regularly cycling women <38 years of age. Immature oocytes were aspirated transvaginally on cycle day 8-9 after priming with FSH (Gonal-F 150 IU/day for 3 days, initiated on day 3). Oocytes were matured in Dyrkningsmedie til IVM supplemented with recombinant FSH (rFSH) 0.075 IU/ml and HCG 0.5 IU/ml for 28-30 h. In group I (n = 63 oocytes obtained from the first 23 cycles) the culture medium was supplemented with 2% (w/v) HSA. In group II (n = 74 oocytes obtained from the following 28 cycles) the medium was supplemented with 10% (v/v) heat-inactivated maternal serum. Intracytoplasmic sperm injection (ICSI) was performed on all methaphase II oocytes. Significantly increased rates of maturation 47/74 (63%) vs. 26/63 (41%) (P < 0.05), pregnancy 6/28 (21%) vs. 0/23 (0%) (P < 0.05) and implantation 6/20 (30%) vs. 0/15 (0%) (P < 0.05) were obtained from oocytes matured in culture medium with maternal serum supplementation compared with oocytes matured in medium supplemented with HSA. These results indicate that factors other than albumin in maternal serum play an important role in maturation and subsequent developmental capacity of human oocytes.

Apoptosis in human cumulus cells in relation to maturation stage and cleavage of the corresponding oocyte.

BACKGROUND: The purpose of this study was to determine the incidence of apoptosis in cumulus cells, and correlate these findings with the maturation stage, fertilization rate and embryo score of the corresponding oocyte, in couples undergoing ICSI due to a male factor. METHODS: The study group consisted of 21 couples where ICSI was performed. The total number of oocyte-cumulus complexes retrieved was 164. Sperm samples were assessed according to the WHO manual, morphology according to the strict criteria and for the presence of apoptosis. The degree of apoptotic DNA fragmentation was determined using the free 3'OH DNA termini in situ with chemically labeled and unlabeled nucleotides. The study was blinded for the technician involved in the assessments of apoptosis in the cumulus cells, apoptosis and morphology in spermatozoa. Ovarian hyperstimulation was carried out according to a long down regulation protocol using GnRH, recFSH and hCG. A maximum of 3 embryos were transferred on day 2 after ICSI. RESULTS: This study demonstrated that the incidence of apoptosis was significantly higher in cumulus cells from germinal vesicle and metaphase I oocytes compared to cumulus cells from metaphase II oocytes (p<0.0001). Non-fertilized metaphase II oocytes showed significantly higher incidence of apoptosis in cumulus cells compared to fertilized metaphase II oocytes (p=0.0082). Furthermore, apoptosis in spermatozoa had an impact on the embryo score (p=0.0087). CONCLUSION: Comparing apoptosis in cumulus cells with maturity of the corresponding oocytes, a significantly higher degree was found related to immature oocytes. Apoptosis in cumulus cells from human metaphase II oocytes impaired the fertilization rate. The degree of fragmentation in the embryo might be correlated to apoptosis in the spermatozoa.

The role of DNA strand breaks in human spermatozoa used for IVF and ICSI.

BACKGROUND: The objective of this study was to determine the incidence of spermatozoa with DNA strand breaks in four clinically different groups of infertile couples, and to correlate DNA damage with other semen analysis parameters, as well as fertilization rates and IVF outcome. METHODS: One group consisted of 75 men where the female partners had a tubal obstruction, Group A. Fifty sperm samples were collected from men in unexplained infertile couples, Group B. Fifty men with oligozoospermia and IVF made up Group C. Finally, 61 men with oligozoospermia and where ICSI was performed made up Group D. Sperm samples were assessed according to the WHO manual and for the presence of DNA strand breaks in spermatozoa. The study was blinded for the technician involved in the assessment of DNA strand breaks. IVF was carried out according to a long down regulation protocol using GnRH, FSH and hCG. Embryos were transferred on day 2 after fertilization with a maximum of three embryos. RESULTS: This study demonstrated a negative correlation between the proportion of spermatozoa having DNA strand breaks and the proportion of oocytes fertilized after IVF (p<0.01). Furthermore, the number of spermatozoa with DNA strand breaks was important for the pregnancy rate in the group of unexplained infertile couples. After ICSI no association was found between spermatozoa with DNA strand breaks and fertilization rates (p>0.05). CONCLUSION: DNA strand breaks in human spermatozoa impairs fertilization in both unexplained infertile couples and those with oligozoospermia and IVF. However, after ICSI, this impact of DNA strand breaks were not seen. This creates a specific indication and treatment for this new diagnosed group of otherwise unexplained infertile men.

DNA strand breaks in human spermatozoa: correlation with fertilization in vitro in oligozoospermic men and in men with unexplained infertility.

BACKGROUND: The purpose of this study was to examine the correlation between DNA strand breaks in human spermatozoa and semen quality, fertilization rate and IVF outcome. METHODS: A total of 50 men suffering from unexplained infertility and 50 men with oligozoospermia undergoing IVF treatment entered a prospective study. Sperm samples were assessed according to the WHO manual and for the presence of DNA strand breaks in spermatozoa. The study was blinded for the technician involved in assessment of DNA strand breaks. IVF was carried out according to a long down regulation protocol using GnRH, FSH and hCG. The ova were inseminated with 200,000 spermatozoa/ml. Embryos were transferred on day 2 after fertilization with a maximum of three embryos. RESULTS: This study demonstrates a negative correlation between the proportion of spermatozoa having DNA strand breaks and the proportion of oocytes fertilized after IVF. CONCLUSION: The number of human spermatozoa with DNA strand breaks is a good predictor for fertilization rate in couples suffering from unexplained infertility and undergoing IVF treatment.

DNA strand breaks in human spermatozoa: a possible factor, to be considered in couples suffering from unexplained infertility.

BACKGROUND: The aim of this study was to assess the presence of DNA strand breaks and sperm cell morphology in men suffering from unexplained infertility, and to compare these results with normal fertile and oligozoospermic men. METHODS: One fresh sperm sample from proven fertile sperm donors (n=20) and from infertile men with oligozoospermia, (<20 x 10(6)/ml sperm cells) (n=74), and men suffering from unexplained infertility (>20 x 10(6)/ml sperm cells) (n=39) who delivered two sperm samples with 24 hours interval, were tested for the presence of DNA strand breaks in the spermatozoa by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. Correlations to other sperm parameters, sperm cell counts, motility, activation and Krüger's strict criteria were performed. RESULTS: DNA strand breaks in sperm cell nuclei were found significantly more often in sperm samples from men suffering from unexplained infertility compared to those from normal fertile men, and significantly more rarely compared with sperm samples from men with oligozoospermia. The percentages of normal spermatozoa (Krüger's strict criteria) were significantly lower in samples from men suffering from unexplained infertility compared to those of normal fertile men, but significantly higher compared to those of men with oligozoospermia. No difference was found between first and second day samples used for insemination, as regards DNA strand breaks, sperm cell morphology, total number of motile sperm cells, activation and motility degree. CONCLUSION: The present data suggest that a subgroup of men suffering from unexplained infertility have DNA strand breaks in their sperm cell DNA. This group might suffer from the same malfunction as many men with oligozoospermia, however, their apoptotic activated sites in the testis are different. Delivery of sperm samples with 24 hours interval does not affect any sperm cell counts, CASA, DNA strand breaks or morphology findings in sperm samples from men suffering from unexplained infertility.

Sperm morphology and IVF: embryo quality in relation to sperm morphology following the WHO and Krüger's strict criteria.

BACKGROUND: The aim of this study was to examine the correlation between sperm morphology and embryo quality/IVF outcome. METHODS: The implication of sperm morphology assessment before an IVF cycle was evaluated. A total of 100 IVF couples where the female partner had either tubal factor (n=50) or unexplained infertility (n= 50) entered a prospective study, and sperm samples for the actual cycle were assessed according to the strict criteria and WHO criteria. The study was blinded for the technician involved in sperm morphology analyzing. IVF was carried out according to a long down regulation protocol using GnRH/FSH/hCG and ova were inseminated with 200,000 spermatozoa/ml. Embryos were transferred on day 2 post fertilization in a maximum of three embryos. RESULTS: No significant differences were found between the groups regarding age of the female partner (mean=34.3), no. oocytes retrieved (mean=8.5), fertilization (66.5%), pregnancies (pos. S-hCG/transfer 39.6%) or 'Take home baby rate' (birth rate/transfer 30.0%). As to the score of Krüger's strict criteria and the WHO criteria, we found no correlation between this score and cleavage rate, embryo development or pregnancies. The WHO criteria were found to be a better predictor for fertilization rate than the Krüger's criteria (p<0.002). CONCLUSION: The strict criteria or sperm evaluation according to WHO have no better predictive value for the outcome of routine IVF.

DNA strand breaks in human sperm cells: a comparison between men with normal and oligozoospermic sperm samples.

BACKGROUND: The aim of this report was to compare the degree of DNA strand breaks in known normal fertile men to those with oligozoospermia, and evaluate the presence of DNA strand breaks in normal raw sperm, after Percoll and swim-up and following conventional cryopreservation, as all these preparation methods might differ in selection of healthy sperm cells. METHODS: Sperm samples from proven fertile sperm donors (n=20) and infertile men with oligozoospermia (n=33) were tested for the presence of DNA strand breaks in the spermatozoa, by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. A correlation to other sperm parameters, sperm counts, motility and Krüger's strict criteria was performed. RESULTS: DNA strand breaks were found significantly more often in sperm samples from men with oligozoospermia compared to sperm samples of normal fertile men. The degree of spermatozoa with DNA strand breaks was correlated proportional with the degree of morphological pathological sperm cells judged by Krüger's strict criteria. The percentage of spermatozoa with DNA strand breaks in the samples was not influenced by procedures such as the swim-up technique, Percoll gradients or cryopreservation and thawing. CONCLUSION: DNA strand breaks were found significantly more often in men with oligozoospermia compared to normospermic men. The DNA strand breaks might play an important role for the maturation process of the spermatozoa in the same way as apoptosis is controlling the number of early meiotic germ cells in the testis, and hereby play an important role in advanced fertility treatments (ICSI).