Developmental dynamics of IMSI-derived embryos: a time-lapse prospective study.

Because sperm vacuoles were marked as zones without chromatin in the sperm nucleus, which may reflect underlying chromosomal or DNA defects, this study considered whether they influence the morphology and dynamics of early developmental events in preimplantation embryos. Oocytes were injected with spermatozoa of four classes, according to the number and size of vacuoles at ×6000 magnification, and derived embryos were observed under time-lapse microscopy. For each embryo, the times of pronuclei appearance and disappearance and the first, second and third divisions were determined and related to its respective class of injected spermatozoa and its developmental stage. Embryos arising from normal class-I spermatozoa (without vacuoles) reached the 4-cell stage significantly earlier than embryos developed from class-IV spermatozoa (with large vacuoles and other abnormalities) (P=0.012). Blastocysts from class-I spermatozoa required the shortest mean time for all developmental events in comparison with blastocysts from spermatozoa of other classes (with vacuoles). Blastocysts also showed significantly earlier first division than arrested embryos in embryos arising from class-I spermatozoa (P=0.033). An insight into the developmental dynamics of embryo development according to morphology and head vacuoles of injected spermatozoa in morphologically selected sperm-derived embryos was observed for the first time.

Intracytoplasmic morphologically selected sperm injection improves development and quality of preimplantation embryos in teratozoospermia patients.

This prospective randomized study investigated whether intracytoplasmic sperm injection (ICSI) outcome can be improved with sperm preselection under ×6000 magnification and intracytoplasmic morphologically selected sperm injection (IMSI) in patients with teratozoospermia and characterized embryo development and quality regarding sperm morphology and presence of head vacuoles. Couples with isolated teratozoospermia were divided into two groups: IMSI group (n=52) and ICSI group (n=70) and fertilization, blastocyst and clinical pregnancy rates were compared. Oocytes from 30 randomly chosen patients from the IMSI group were injected with spermatozoa that had been previously classified under ×6000 magnification into four classes according to the number and size of vacuoles in the head and then cultured separately. Pronuclear morphology, embryo development and blastomere viability were estimated to investigate the influence of sperm morphology, especially vacuoles, on embryo developmental capacity. A significantly higher clinical pregnancy rate was achieved in the IMSI group compared with the ICSI group (48% versus 24%, P<0.05). Fertilization with spermatozoa without head vacuoles yielded higher number of morphologically normal zygotes, higher blastocyst rate and smaller proportion of arrested embryos than spermatozoa with vacuoles and other head defects. IMSI is a method of choice in patients with teratozoospermia.

Slow oocyte freezing and thawing in couples with no sperm or an insufficient number of sperm on the day of in vitro fertilization.

BACKGROUND: The clinical results of in vitro fertilization of slowly frozen-thawed oocytes are known to be significantly worse than those obtained by fresh oocytes. Little is known about the factors affecting the clinical outcome of frozen-thawed oocytes. The aim of this retrospective study was to explore the role of oocyte cryopreservation in the group of patients with no available sperm on the day of in vitro fertilization. Additionally, the effects of the female serum FSH level and sperm quality on the clinical outcome of frozen-thawed oocytes were evaluated. METHODS: Oocytes were slowly frozen and thawed in 22 infertile couples with no sperm or insufficient number of sperm on the day of in vitro fertilization (IVF). In 9 couples with severe azoospermia or oligoasthenoteratozoospermia frozen-thawed oocytes were fertilized by autologous sperm of bad quality when available (Group 1). In 13 couples with non-ejaculation due to psychological stress on the day of classical IVF or severe azoospermia frozen-thawed oocytes were fertilized by autologous or donated sperm of normal quality (Group 2). Oocytes were thawed in 23 cycles and microinjected by the autologous or donated sperm, when available. The clinical outcome of intracytoplasmic sperm injection--ICSI (fertilization, blastocyst, and pregnancy rates) was compared to the outcome of fresh oocytes of the same group of patients; additionally, the female serum FSH level and the sperm quality were compared. RESULTS: In all couples, 70.5% of oocytes survived the freeze-thaw procedure. After ICSI, 61.5% of thawed oocytes were fertilized. Twenty one% of embryos developed to the blastocyst stage. The pregnancy rates per embryo transfer and freeze-thaw cycle were 33.3% and 17.4%, respectively. All pregnancies ended in the birth of a baby without congenital anomalies. In patients with severe azoospermia or oligoasthenoteratozoospermia there was no statistically significant difference in pregnancy rates per cycle obtained by thawed oocytes vs. fresh oocytes in previous ICSI cycles (14.2% vs. 13.6%) but there was a higher proportion of abnormal, non-cleaved or triploid zygotes when frozen-thawed oocytes were microinjected (33.3% vs. 11.8%; P < 0.01). The female serum FSH levels did not affect the survival and fertilization of frozen-thawed oocytes, but in patients with increased serum FSH level no pregnancies were achieved. After the complete freeze-thaw cycle, there was a significantly higher fertilization rate and tendency to higher pregnancy rates per thawing cycle after the microinjection of autologous or donated sperm of normal quality than autologous sperm of poor quality. CONCLUSION: The slow oocyte freezing and thawing is a valuable method when no or insufficient number of sperm are available on the day of in vitro fertilization. The quality of sperm is an important factor affecting the clinical outcome achieved by frozen-thawed oocytes.

The IMSI procedure improves poor embryo development in the same infertile couples with poor semen quality: a comparative prospective randomized study.

BACKGROUND: Sperm of poor quality can negatively affect embryo development to the blastocyst stage. The aim of this comparative prospective randomized study was to evaluate the role of an intracytoplasmic morphologically selected sperm injection (IMSI) in the same infertile couples included in the programme of intracytoplasmic sperm injection (ICSI) due to their indications of male infertility which had resulted in all arrested embryos following a prolonged 5-day culture in previous ICSI cycles. METHODS: Couples exhibiting poor semen quality and with all arrested embryos following a prolonged 5-day culture in previous ICSI cycles were divided into two groups: Group 1: IMSI group (n = 20) with IMSI performed in a current attempt and Group 2: ICSI group (n = 37) with a conventional ICSI procedure performed in a current attempt of in vitro fertilization. Fertilization rate, embryo development, implantation, pregnancy and abortion rates were compared between current IMSI and conventional ICSI procedures, and with previous ICSI attempts. RESULTS: The IMSI group was characterized by a higher number of blastocysts per cycle than the ICSI group (0.80 vs. 0.65) after a prolonged 5-day embryo culture. There was a significantly lower number of cycles with all arrested embryos and cycles with no embryo transfer in the IMSI group versus the ICSI group (0% vs. 27.0%, p = 0.048). After the transfer of embryos at the blastocyst or morula stage (on luteal day 5) a tendency toward higher implantation and pregnancy rates per cycle was achieved in the IMSI group compared to the ICSI group (17.1% vs. 6.8%; 25.0% vs. 8.1%, respectively), although not statistically significant. After IMSI, all pregnancies achieved by the blastocyst transfer were normally on-going, whereas after ICSI, two of three pregnancies ended in spontaneous abortion. After IMSI, two pregnancies were also achieved by the morula stage embryos, whereas after the conventional ICSI procedure, embryos at the morula stage did not implant. CONCLUSIONS: The IMSI procedure improved embryo development and the laboratory and clinical outcomes of sperm microinjection in the same infertile couples with male infertility and poor embryo development over the previous ICSI attempts.

Successful freezing and thawing of blastocysts cultured in sequential media using a modified method.

OBJECTIVE: To evaluate the clinical role of blastocyst freezing and thawing after prolonged culturing in sequential media. DESIGN: Retrospective analysis of 293 blastocyst freeze-thawing cycles. SETTING: University hospital infertility unit. PATIENT(S): Nonselected couples undergoing IVF. INTERVENTION(S): Blastocysts were frozen and thawed by a modified method. MAIN OUTCOME MEASURES: Blastocyst recovery after freeze-thawing and pregnancy rates after the transfer. Evaluation of the effect of the number of transferred blastocysts, the method of IVF, and of the woman's age on the results achieved by frozen-thawed blastocysts. RESULT(S): Frozen-thawed blastocysts provided a 29.5% clinical pregnancy rate per transfer. After the transfer of three blastocysts the pregnancy rate was 42.0%, and after the transfer of one or two blastocysts it was approximately the same (25.0% and 28.0%, respectively). The method of IVF did not affect pregnancy rates, but the increasing age of the woman did. Pregnancies were characterized by a low abortion rate (8.0%) regardless of the age of the woman. CONCLUSION(S): A modified method for blastocyst freeze-thawing provides good clinical results. It offers the possibility for a single-thawed blastocyst transfer and represents a good alternative for older women because of its lower risk of spontaneous abortion.

Recombinant FSH vs. urinary FSH for ovarian stimulation in in vitro fertilization.

OBJECTIVE: To compare ovarian stimulation with recombinant FSH (rFSH) vs. urinary FSH (uFSH) in terms of hormonal events within ovarian follicles and the outcome of in vitro fertilization. STUDY DESIGN: A prospective randomized comparative study of rFSH (n = 70) vs. uFSH (n = 61) ovarian stimulation. Hormone determinations were serum estradiol (E2) on the day of human chorionic gonadotropin (hCG) administration, and E2, androstenedione (A) and testosterone (T) at the time of follicular aspiration in the follicular fluid and serum. RESULTS: The total dose of gonadotropins required and the length of ovarian stimulation were the same in the 2 groups. In follicular fluid the E2 and the A levels were significantly higher in the rFSH group (3,065 +/- 1,646 vs. 2,368 +/- 1,240 nmol/L, P = .004, and 103.7 +/- 51.6 vs. 89.0 +/- 42.3 nmol/L, P = .042, respectively), whereas A:E2 and T:E2 ratios were significantly lower (39.6 +/- 22.5 vs. 52.3 +/- 59.6, P = .042, and 9.1 +/- 4.7 vs. 17.6 +/- 26.9, P = .006, respectively). Serum hormonal levels, number of oocytes retrieved and pregnancy rates did not differ significantly between the groups. CONCLUSION: rFSH provides results similar to those of uFSH. rFSH enhances steroidogenesis and provokes different androgen/estrogen ratios than does uFSH without influencing the outcome of in vitro fertilization.

Gene expression profiling of human oocytes developed and matured in vivo or in vitro.

The quality of the human oocyte determines the success of fertilization and affects the consequent embryo development, pregnancy and birth; it therefore serves as a basis for human reproduction and fertility. The possibility to evaluate oocyte quality in the in vitro fertilization programme is very limited. The only criterion which is commonly used to evaluate oocyte quality is its morphology. There is a mass of oocytes in the in vitro fertilization programme which are not fertilized in spite of normal morphology. In the past, several attempts focused on oocyte gene expression profiling by different approaches. The results elucidated groups of genes related to the human oocyte. It was confirmed that some factors, such as oocyte in vitro maturation, are detectable at the molecular level of human oocytes and their polar bodies in terms of gene expression profile. Furthermore, the first genetic evaluations of oocyte-like cells developed in vitro from human stem cells of different origin were performed showing that these cells express some genes related to oocytes. All these findings provide some new knowledge and clearer insights into oocyte quality and oogenesis that might be introduced into clinical practice in the future.

Single fresh and frozen-thawed blastocyst transfer using hyaluronan-rich transfer medium.

The purpose of this prospective randomized study was to evaluate use of a hyaluronan-rich transfer medium in fresh and frozen-thawed single blastocyst transfer. The study included 279 single blastocyst transfers in women aged<37 years in their first, second or third treatment cycle. According to the type of single blastocyst transfer (fresh elective or frozen-thawed) the women were divided into two study and two control groups. In both study groups (n=130) transfers were performed using hyaluronan and in the control groups (n=149) a conventional transfer medium was used. The results indicate that fresh elective single blastocyst transfer with hyaluronan results in significantly higher pregnancy rates in a selected subgroup of women; those with >or=2 blastocysts developed to day 5 and a previous implantation failure (55% versus 10%; P=0.012). Overall pregnancy rates after fresh elective and frozen-thawed single blastocyst transfer were similar in both study and control groups.

Outcome of frozen-thawed blastocysts derived from gonadotropin releasing hormone agonist or antagonist cycles.

PURPOSE: The aim of this retrospective study was to compare the outcome of frozen-thawed blastocysts derived from the cycles using controlled ovarian stimulation with GnRH agonists vs. GnRH antagonists. METHODS: Survival, pregnancy and cumulative live birth rates in 231 freeze-thaw cycles derived from the GnRH agonist cycles (GnRH agonist group), and in 175 freeze-thaw cycles derived from the GnRH antagonist cycles (GnRH antagonist group) were compared. RESULTS: In the GnRH agonist group significantly higher proportion of blastocysts survived the thawing procedure than in the GnRH antagonist group (86.1% versus 78.5%; p < 0.01). The differences in cumulative live birth rates did not differ significantly between the groups: in the GnRH agonist group the cumulative live birth rate was 16.5%, and in the GnRH antagonist group it was 14.2%. CONCLUSIONS: Frozen-thawed blastocysts derived from the GnRH agonist cycles have better survival rates and similar cumulative live birth rates than those derived from the GnRH antagonist cycles.

Increased ammonium in culture medium reduces the development of human embryos to the blastocyst stage.

The objective of this prospective study was to evaluate the effect of ammonium accumulated in sequential media and determined by enzymatic spectrophotometric method on the blastocyst development in 281 human embryos from 100 stimulated and natural in vitro fertilization (IVF) cycles. Ammonium concentration was increased in 62% of cycles and was correlated negatively with the blastocyst development after classical IVF, but not after intracytoplasmic sperm injection (ICSI).

Sperm single-stranded DNA, detected by acridine orange staining, reduces fertilization and quality of ICSI-derived embryos.

PURPOSE: The aim of this study was to evaluate the effect of sperm single-stranded DNA, detected by acridine orange (AO), and classical sperm parameters on embryonic quality after ICSI. METHODS: Before ICSI, the spermatozoa of 183 infertile patients with oligo-, astheno-, teratozoospermia (n = 147), or more than one previous unsuccessful conventional IVF attempt (n = 36) were stained by AO to assess the presence of single-stranded DNA. Two days after ICSI, the embryos of 135 patients were scored for morphology, fragmentation included. Embryos of 48 couples were cultured for 4 days to develop to the morula or blastocyst stage. At most 2 embryos were transferred on Day 2 or 4. RESULTS: When the level of spermatozoa with single-stranded DNA was increased, there was a significantly lower fertilization rate after ICSI. Besides, increased sperm single-stranded DNA resulted in a higher proportion of heavily fragmented embryos on Day 2 (P < 0.05). In patients with an increased level of spermatozoa with single-stranded DNA, a significantly higher number of embryos were arrested in spite of prolonged culturing (P < 0.05). Classical sperm parameters did not affect the quality and developmental potential of ICSI-derived embryos. No correlation was found between the level of spermatozoa with single-stranded DNA, pregnancy rate, and live-birth rate achieved by ICSI, except in patients with 0% of spermatozoa with single-stranded DNA, in whom the pregnancy rate was significantly higher. CONCLUSIONS: Sperm single-stranded DNA provides additional data on sperm functional capacity in terms of fertilization and embryonic quality after ICSI.