Effect of oocyte donor stimulation on recipient outcomes: data from a US national donor oocyte bank.

STUDY QUESTION: How does ovarian stimulation in an oocyte donor affect the IVF cycle and obstetric outcomes in recipients? SUMMARY ANSWER: Higher donor oocyte yields may affect the proportion of usable embryos but do not affect live birth delivery rate or obstetric outcomes in oocyte recipients. WHAT IS KNOWN ALREADY: In autologous oocyte fresh IVF cycles, the highest live birth delivery rates occur when ~15-25 oocytes are retrieved, with a decline thereafter, perhaps due to the hormone milieu, with super-physiologic estrogen levels. There are scant data in donor oocyte cycles, wherein the oocyte environment is separated from the uterine environment. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study from 2008 to 2015 of 350 oocyte donors who underwent a total of 553 ovarian stimulations and oocyte retrievals. The oocytes were vitrified and then distributed to 989 recipients who had 1745 embryo transfers. The primary outcome was live birth delivery rate, defined as the number of deliveries that resulted in at least one live birth per embryo transfer cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included oocyte donors and recipients at a donor oocyte bank, in collaboration with an academic reproductive endocrinology division. Donors with polycystic ovary syndrome and recipients who used gestational carriers were excluded. The donors all underwent conventional ovarian stimulation using antagonist protocols. None of the embryos underwent pre-implantation genetic testing. The average (mean) number of embryos transferred to recipients was 1.4 (range 1-3). MAIN RESULTS AND THE ROLE OF CHANCE: Per ovarian stimulation cycle, the median number of oocytes retrieved was 30 (range: 9-95). Among the 1745 embryo transfer cycles, 856 of the cycles resulted in a live birth (49.1%). There were no associations between donor oocyte yield and probability of live birth, adjusting for donor age, BMI, race/ethnicity and retrieval year. The results were similar when analyzing by mature oocytes. Although donors with more oocytes retrieved had a higher number of developed embryos overall, there was a relatively lower percentage of usable embryos per oocyte warmed following fertilization and culture. In our model for the average donor in the data set, holding all variables constant, for each additional five oocytes retrieved, there was a 4% (95% CI 1%, 7%) lower odds of fertilization and 5% (95% CI 2%, 7%) lower odds of having a usable embryo per oocyte warmed. There were no associations between donor oocyte yield and risk of preterm delivery (<37 weeks gestation) and low birthweight (<2500 g) among singleton infants. LIMITATIONS, REASONS FOR CAUTION: Ovarian stimulation was exclusively performed in oocyte donors. This was a retrospective study design, and we were therefore unable to ensure proportional exposure groups. These findings may not generalizable to older or less healthy women who may be vitrifying oocytes for planned fertility delay. There remain significant risks to aggressive ovarian stimulation, including ovarian hyperstimulation. In addition, long-term health outcomes of extreme ovarian stimulation are lacking. Lastly, we did not collect progesterone levels and are unable to evaluate the impact of rising progesterone on outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Live birth delivery rates remain high with varying amounts of oocytes retrieved in this donor oocyte model. In a vitrified oocyte bank setting, where oocytes are typically sent as a limited number cohort, recipients are not affected by oocyte yields. STUDY FUNDING/COMPETING INTEREST(S): Additional REDCap grant support at Emory was provided through UL1 TR000424. Dr. Audrey Gaskins was supported in part by a career development award from the NIEHS (R00ES026648).

Vitrification in assisted reproduction: myths, mistakes, disbeliefs and confusion.

The purpose of this work is to update embryologists and clinicians on different approaches in human oocyte and embryo cryopreservation, by clarifying some misunderstandings and explaining the underlying reasons for controversial opinions. The work is based on literature review and critical analysis of published papers or conference abstracts during the last 24 years, with special focus on the last 3 years. Due to the latest advancements in techniques, cryopreservation now offers new perspectives along with solutions to many demanding problems, and has developed from a backup procedure to a successful alternative that is an indispensable constituent of assisted reproductive techniques. However, this progress is not free from controversies, at some points is rather serendipitous, and many factors, including human ones, hamper the selection and widespread application of the most efficient technique for the given task. A better understanding of the basic features of the two rival approaches (slow-rate freezing and vitrification), a clarification of terms and technical details, and a balanced, pragmatic evaluation of possible risks and potential, or definite, gains are required to accelerate advancement. Alternatively, the increasing flow of patients to the few assisted reproduction clinics and countries that are highly successful in this field will enforce the required changes in methodology and mentality worldwide.

Co-transfer of embryos derived from cryopreserved and fresh natural cycle oocytes: a pilot study.

Italian legislation regarding reproductive medicine prohibits embryo storage while allowing cryopreservation of supernumerary oocytes. This study evaluated the effect of fresh oocytes obtained from natural unstimulated cycles on the clinical success rates derived from the use of frozen-thawed (FR-TH) oocytes obtained following ovarian stimulation. For 36 women, intracytoplasmic sperm injection was performed on FR-TH oocytes supplemented by a fresh oocyte, if available, derived from a natural cycle in which gonadotrophin-releasing hormone-antagonist was used for premature LH surge control. The retrieval rate of fresh oocytes was 61.1% and survival rate of FR-TH oocytes was 43.6%. The fertilization rate of fresh and FR-TH oocytes was 70% and 52.5%, respectively. Fifty embryos were transferred, 14 of them developed from fresh oocytes and 36 from FR-TH oocytes. Six pregnancies occurred in 10 cycles in which the embryos developed from fresh and FR-TH oocytes (pregnancy rate 60.0%) and two in 12 patients in whom the embryos were obtained from only FR-TH oocytes (pregnancy rate 16.7%) (P < 0.05). In summary, the data demonstrate that the transfer of embryos derived from oocytes cryopreserved following a previous ovarian stimulation and an embryo developed from a fresh one retrieved in natural cycle ensures an excellent clinical outcome.

Complementation of HLA-DQA and -DQB genes confers susceptibility and protection to insulin-dependent diabetes mellitus.

Lack of an aspartic acid 57 in the HLA-DQ beta chain was introduced as a genetic marker of insulin-dependent diabetes mellitus (IDDM). Because 25% of the control population carries the same marker, we analyzed the DQ locus for the presence of more specific disease susceptibility markers, taking into account a possible role for the polymorphic DQA gene. We thereby identified the DQA3-DQB3.2/DQA4.1-DQB2 (DQA1*0301-DQB1*0302/DQA1*0501-DQB1*0201) genotype which was detected in 30% of the 268 typed IDDM patients and only in 1% of the 331 typed healthy controls, resulting in a relative risk of 35. This genetic marker was more frequent in patients with clinical onset before age 18 years (36%) than in patients diagnosed between age 18 and 40 years (22%) and was not observed in patients with non-IDDM. The new susceptibility genotype DQA3-DQB3.2/DQA4.1-DQB2 (DQA1*0301-DQB1*0302/DQA1*0501-DQB1*0201) may explain the well-known excess of DR3/DR4 heterozygous IDDM patients and is expected to help identify individuals at risk for developing the disease.

Sperm centriole disfunction and sperm immotility.

The two main causes of complete or nearly complete asthenozoospermia are necrozoospermia (presence of only non-viable spermatozoa) and the different ultrastructural abnormalities of spermatozoa. Ultrastructural alterations may affect also the function of the sperm centrosome, which can result in impaired motility. Because in human the inheritance of the centrosome is paternal and thus linked to the sperm, morphological or functional alterations of it can also be associated with fertilization abnormalities of the oocyte and cleavage irregularities of the embryo. Most of the cases of asthenozoospermia can be treated efficiently by intracytoplasmic sperm injection (ICSI) using ejaculated sperm (from repeated ejaculation) in combination with hypo-osmotic swelling test (HOST) or using testicular sperm depending on the etiology of the impairment. Replacement of abnormal centriole using donor sperm is a theoretical possibility, but at present it is not an efficient method.

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.

Prospective, auto-controlled study on reinsemination of failed-fertilized oocytes by intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the possible influence of late fertilization after standard IVF on the results of reinsemination of assumed failed-fertilized oocytes by microinjection and to examine the correlation between the effect of aging of (failed-fertilized) oocytes and the ability of these oocytes to become fertilized. DESIGN: Trial 1: Group 1 (injected-day 1), 93 failed-fertilized oocytes injected 1 day after ovum pick-up; group 2 (control), 82 failed-fertilized oocytes with no microinjection performed. Trial 2: Group 1 (ICSI-day 1), 40 failed-fertilized oocytes injected 1 day after ovum pick-up; group-2 (ICSI-day 2), 40 failed-fertilized oocytes injected 2 days after ovum pick-up. In addition, 35 two- to eight-cell stage embryos, obtained after ICSI of IVF failed-fertilized oocytes, were fixed for cytogenetic analysis. MAIN OUTCOME MEASURES: Normal and abnormal fertilization and embryo development. RESULTS: Trial 1: 53% normal (2 pronuclear [PN]) and 25% abnormal (> or = 3PN) fertilization rates were obtained in group 1 (injected-day 1), and 71% of the 2PN and 74% of the > or = 3PN oocytes cleaved with < 50% fragmentation. No pronuclear (> or = 2PN) development occurred in the control group. Trial 2: 45% and 8% normal and 25% and 40% abnormal fertilization rates were obtained, respectively, after ICSI of 1-day-old and 2-day-old failed-fertilized oocytes. Two days after microinjection, 67% and 67% of the 2PN and 80% and 44% of the > or = 3PN oocytes cleaved with < 50% fragmentation in group ICSI-day 1 and in group ICSI-day 2, respectively. CONCLUSIONS: Late fertilization after initial in vitro insemination does not play a role in the high fertilization rate obtained after reinsemination of assumed failed-fertilized oocytes by ICSI. Normal (2PN) fertilization rate, however, decreases strongly and the abnormal (> or = 3PN) fertilization rate increases with oocyte aging and derived embryos seem to have a high incidence of cytogenetic abnormalities.

Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report.

OBJECTIVE: To report a normal pregnancy and the delivery of a healthy child after the combination of in vitro maturation of germinal-vesicle stage oocytes and intracytoplasmic sperm injection (ICSI) in a patient. SETTING: Procedures were performed in a tertiary IVF center coupled with an institutional research environment. MAIN OUTCOME MEASURES: Maturation rate of immature oocytes after in vitro maturation and intactness, fertilization, and developmental rates of oocytes after microinjection. RESULTS: Nine of 14 germinal-vesicle stage oocytes matured to the metaphase II stage after 30 hours of in vitro culture (64%). Seven of eight injected and intact oocytes fertilized normally (78%) and five of them cleaved with < 20% fragmentation (71%). Four embryos were transferred and a singleton pregnancy was obtained that ended in the delivery of a healthy child. CONCLUSION: In vitro maturation of immature oocytes together with ICSI can result in normal fertilization, embryo development, pregnancy, and the delivery of healthy child.

An improved treatment procedure for testicular biopsy specimens offers more efficient sperm recovery: case series.

OBJECTIVE: To report an improved sperm recovery procedure from testicular biopsy specimens for intracytoplasmic sperm injection (ICSI). DESIGN: Case series and controlled study. SETTING: Procedures were performed in a tertiary IVF center. PATIENT(S): Nonobstructive azoospermic cases (15 patients) and obstructive azoospermic cases (5 patients). INTERVENTION(S): Intracytoplasmic sperm injection was carried out using testicular sperm isolated from a testicular biopsy specimen either with or without erythrocyte lysing buffer treatment. MAIN OUTCOME MEASURE(S): The time required to collect spermatozoa and the intactness and fertilization and developmental rates of oocytes. RESULT(S): In 7 of the 15 nonobstructive cases, it was possible to perform ICSI when, after shredding of the testicular tissue, no (or virtually no) sperm were present. There was no difference in the fertilization rates (83% and 68%) and developmental rates (87% and 89%) of the 54 sibling oocytes from another 5 patients in whom ICSI was carried out with sperm either treated or not treated with erythrocyte lysing buffer. CONCLUSION(S): Erythrocyte lysing buffer treatment of testicular biopsy specimens enhances the efficiency of sperm collection in those cycles in which spermatozoa are present and does not affect fertilization and embryo development.

The predictive value of uterine artery blood flow measurements for uterine receptivity in an intracytoplasmic sperm injection program.

OBJECTIVE: To assess whether uterine artery blood flow impedance, measured as the pulsatility index on the day of ET in patients undergoing IVF-ET with microinjection, can predict the likelihood of pregnancy. DESIGN: Prospective clinical study. SETTING: A tertiary referral center for assisted reproduction. PATIENT(S): Seventy patients undergoing intracytoplasmic sperm injection (ICSI) for andrologic indications. INTERVENTION(S): Transvaginal color Doppler examination performed on the day of ET. MAIN OUTCOME MEASURE(S): Mean (+/- SD) pulsatility index value of the left and right uterine arteries, serum E2 levels, implantation rates, and ongoing pregnancy rates (PRs). RESULT(S): The patients were divided into pregnant and nonpregnant groups and were separated according to whether the pulsatility index was low (1.00-1.99), medium (2.00-2.99), or high (> or = 3.00). The pulsatility index values did not change statistically in the pregnant and nonpregnant groups. The implantation rates were 19.5%, 15.4%, and 25% for the low-, medium-, and high-pulsatility index groups, respectively. The ongoing PRs for the same groups were 35.3%, 26.7%, and 37.5%, respectively. CONCLUSION(S): The study suggests that blood flow, measured as the pulsatility index on the day of ET, cannot predict the likelihood of pregnancy in stimulated cycles of ICSI.

[Dynamic MR examination of the breast--histological correlations]. / Az emló dinamikus MR-vizsgálata--szövettani korreláció.

The authors outline dynamic MR mammography (dMRM) as a highly sensitive diagnostic method for the examination of the breast. In a retrospective study relating to 84 processed cases, in the knowledge of the cytological-histological findings the diagnostic accuracy of the examinations was determined. The role of the method in detecting benign and malignant changes of the breast has been estimated. Misdiagnosed cases have been analysed and recommendations for the application of the method are included. The MR proved to be positive in 32 cases and negative in 3 cases of the analysed 35 malignant tumors. Benign lesions were found at microscopy in 49 cases, of which MR correctly diagnosed 40. The sensitivity and the specificity of dynamic MR mammography were 91% and 82%.

A review of the promises and pitfalls of oocyte and embryo metabolomics.

Embryo viability assessment is one of the most important and challenging tasks in IVF. Evaluation of embryo quality is critical when selecting the best embryo(s) to transfer or cryopreserve. Until recently, the only instrument used for embryo evaluation was the inverted light microscope, which provided information based on morphological characteristics. Developmental and morphological information gained from microscopic assessment have been positively associated with IVF outcomes, including pregnancy and implantation rates. However, based on general statistics, it is clear that IVF currently still results in relatively low pregnancy rates, while simultaneously being associated with relatively high multiple implantation rates. Only with novel embryo assessment and selection procedures would it be possible to improve these outcomes. Accordingly, it has been proposed that it is possible to test the culture environment of a developing embryo to gain valuable information regarding its viability. Different approaches have been used. These include the measurement of oxygen consumption by the embryo and testing of the soluble HLA-G in the environment, as it was proposed that secretion of HLA-G is associated with higher implantation rates. Amino acid turnover, which appears to be correlated to blastocyst development, can be measured as an indication of embryo viability. Other approaches, such as time-lapse video observation or cumulus cell gene expression analysis, may be used in the future to gain a broader understanding of embryo viability. Proteomics and metabolomics are also useful tools for assessment of embryo developmental potential. Results from recent studies on predicting embryo viability by analyzing the metabolome of different stage embryos are promising, as increases in pregnancy and implantation rates were obtained using the metabolomic profile for embryo selection. Several novel approaches are currently being developed to aid in viability assessment. These need to be evaluated in prospective clinical trials, while considering their practicality in the clinical laboratory.

A highly immunogenic trivalent T cell receptor peptide vaccine for multiple sclerosis.

BACKGROUND: T cell receptor (TCR) peptide vaccination is a novel approach to treating multiple sclerosis (MS). The low immunogenicity of previous vaccines has hindered the development of TCR peptide vaccination for MS. OBJECTIVE: To compare the immunogenicity of intramuscular injections of TCR BV5S2, BV6S5 and BV13S1 CDR2 peptides in incomplete Freunds adjuvant (IFA) with intradermal injections of the same peptides without IFA. METHODS: MS subjects were randomized to receive TCR peptides/IFA, TCR peptides/saline or IFA alone. Subjects were on study for 24 weeks. RESULTS: The TCR peptides/IFA vaccine induced vigorous T cell responses in 100% of subjects completing the 24-week study (9/9) compared with only 20% (2/10) of those receiving the TCR peptides/saline vaccine (P =0.001). IFA alone induced a weak response in only one of five subjects. Aside from injection site reactions, there were no significant adverse events attributable to the treatment. CONCLUSIONS: The trivalent TCR peptide in IFA vaccine represents a significant improvement in immunogenicity over previous TCR peptide vaccines and warrants investigation of its ability to treat MS.

Chemically and mechanically induced membrane fusion: non-activating methods for nuclear transfer in mature human oocytes.

Most current studies of nuclear transfer in mammalian oocytes have used electrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplasts. However, the application of electrofusion to human oocytes is hampered by the relative ease with which this procedure induces oocyte activation. Here we tested a previously described chemical fusion technique and an original mechanical fusion procedure in this application. Enucleated metaphase II oocytes were first agglutinated with karyoplasts originating from other metaphase II oocytes and then induced to fuse with the use of polyethylene glycol or by micromanipulation with an intracytoplasmic sperm injection (ICSI) micropipette. Both techniques yielded a high frequency of fusion and did not cause oocyte activation. Moreover, the reconstructed oocytes were easily activated by subsequent treatment with ionophore A23187 and 6-dimethylaminopurine. These techniques may be used in attempts to alleviate female infertility due to insufficiency of ooplasmic factors by nuclear transfer from patients' oocytes to enucleated donor oocyte cytoplasts. For eventual future use in human cloning, they would ensure prolonged exposure of transferred nuclei to metaphase promoting factor, which appears to be required for optimal nuclear reprogramming.

Maturation of human cumulus-free germinal vesicle-stage oocytes to metaphase II by coculture with monolayer Vero cells.

The objective of this study was to evaluate and compare the efficacy of coculture with Vero cells (monkey kidney cells) and in-vitro culture in B2 Ménézo medium for the maturation of germinal vesicle (GV)-stage cumulus-free human oocytes to metaphase II. Cumulus-free human GV-stage oocytes obtained after ovulation induction and retrieval via vaginal ultrasound echo-guided puncture were put either in coculture with Vero cells or in 25 microliters droplets of B2 Ménézo medium. In a controlled randomized study, 145 GV-stage oocytes were matured in vitro: 72 were placed in the coculture system with Vero cells and 73 were cultured in B2 alone. After 30 h of in-vitro culture, 82.4% had dissolved the nuclear membrane and extruded the first polar body in the coculture system compared with 37% in B2 medium (P < 0.001). A significant difference was also noticed in the number of GV-stage oocytes which underwent GV breakdown; the proportion of oocytes which did not extrude the first polar body (metaphase I) was 1.6% in the coculture system compared to 44.0% in the B2 medium. It can be concluded that coculture with Vero cells improves the in-vitro maturation of cumulus-free human GV-stage oocytes.

Simultaneous amplification of the two most frequent mutations of infantile Tay-Sachs disease in single blastomeres.

Tay-Sachs disease is a lysosomal storage disease, which in its most severe form leads inexorably to death during infancy. We have developed a method for preimplantation diagnosis, using polymerase chain reaction (PCR) technology, by which the two most frequent mutations occurring in this disease can be amplified simultaneously. We have tested this method on single blastomeres and have compared four lysis methods: (i) boiling in water at 94 degrees C for 15 or (ii) 30 min, and (iii) incubation in an alkaline lysis buffer for 30 min at 94 degrees C or (iv) at 65 degrees C for 10 min. The amplification percentages were 21, 67, 71 and 91% respectively.

Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection.

Knowledge of the timing of the stages of fertilization in humans is still limited because the time of gamete fusion is not known when pre-ovulatory or in-vitro matured cumulus-enclosed oocytes are inseminated. We therefore studied the morphological nuclear changes in 14 patients' oocytes by means of light microscopic observation at 2, 4, 6, 8, 16, 18 and at 20 h after intracytoplasmic single sperm injection (ICSI). A total of 144 metaphase II oocytes were injected with the spermatozoa of the patients' partners. Out of the 134 oocytes that survived the injection, 93 displayed two pronuclei in the course of the observation period (69%). Out of the 93 normally fertilized oocytes, 21 extruded the second polar body at 2 h after micro-injection (23%) and 63 oocytes at 4 h (68%). Pronuclei appeared as early as 6 h after ICSI in 16 normally fertilized oocytes (17%). At 8 h, 75 (80%) oocytes had two visible pronuclei, at 16 h 92 (99%), at 18 h 76 (82%) and at 20 h 63 (68%). In 24 oocytes (26%) the appearance of pronuclei was asynchronous, while the disappearance of the pronuclei was always synchronous, except in one oocyte. Nine of the 134 successfully injected oocytes showed three equal-sized pronuclei (6.7%). Four of the nine multi-pronucleated oocytes did not extrude the second polar body at all, while the time sequence of appearance of pronuclei was similar to that of the normally fertilized oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Patients with absolutely immotile spermatozoa and intracytoplasmic sperm injection.

The microinjection of completely immotile spermatozoa may impair the outcome of intracytoplasmic sperm injection (ICSI). Eleven couples underwent an initial ICSI cycle with 100% immotile freshly ejaculated spermatozoa. Two-pronuclear fertilization ensued in 18 of 145 (12.4%) successfully injected oocytes. None of these cycles resulted in a pregnancy. Nine couples underwent ICSI in subsequent cycles (n = 16). Ejaculated spermatozoa were injected in 15 cycles and testicular spermatozoa in one cycle. In 10 of the 15 cycles, motile spermatozoa were available at the time of injection. Motile testicular spermatozoa could also be injected. In the subsequent cycles, 91 of 176 (51.7%) successfully injected oocytes fertilized normally and four patients became pregnant. In the subsequent cycles where again immotile spermatozoa had to be injected no pregnancies occurred. In four subsequent cycles embryo cryopreservation was carried out. After replacement of two frozen-thawed embryos one additional pregnancy was obtained. In all, five healthy infants were born. It has been ascertained that motile spermatozoa can be detected either in repeated ejaculates or after testicular biopsy. The causes of total asthenozoospermia are variable and the problem is a sporadic rather than a permanent condition.

Special applications of intracytoplasmic sperm injection: the influence of sperm count, motility, morphology, source and sperm antibody on the outcome of ICSI.

The relationship between the three basic parameters of ejaculated spermatozoa, i.e. concentration, motility and morphology, and the results of intracytoplasmic sperm injection (ICSI) were investigated in 838 microinjection cycles. A further 123 ICSI treatment cycles in which testicular spermatozoa were used for microinjection were also evaluated. The influence of anti-sperm antibodies (ASA) on the outcome of ICSI was investigated by analysing 55 cycles where the proportion of ASA-bound spermatozoa was > or =80%. After microinjection, oocyte intactness, fertilization, embryo cleavage, transfer and pregnancy rates were recorded and compared. The results showed that neither the type nor the extent of sperm impairment had an important influence on the outcome of ICSI when ejaculated spermatozoa were used. Only two very rare conditions had a strongly negative influence on the result of ICSI, i.e. where immotile (presumably dead) spermatozoa or where round-headed spermatozoa were injected into the oocyte. Neither the proportion of ASA-bound spermatozoa, the type of dominantly present ASA, nor the location of ASA on the spermatozoa had an important influence on fertilization, embryo development or pregnancy rates after ICSI. In most of the cycles combined with testicular biopsy (79%), there were enough motile spermatozoa present in the wet preparation for injection of all the oocytes. Injection of motile testicular spermatozoa led to a higher normal fertilization rate than did injection of non-motile spermatozoa (65 versus 21%). It can be concluded that injection of motile (living) spermatozoa into oocytes is the most important factor in determining good results with ICSI and that other sperm parameters do not have a strong influence on the outcome of ICSI.