Clinical outcomes of uninterrupted embryo culture with or without time-lapse-based embryo selection versus interrupted standard culture (SelecTIMO): a three-armed, multicentre, double-blind, randomised controlled trial.

BACKGROUND: Time-lapse monitoring is increasingly used in fertility laboratories to culture and select embryos for transfer. This method is offered to couples with the promise of improving pregnancy chances, even though there is currently insufficient evidence for superior clinical results. We aimed to evaluate whether a potential improvement by time-lapse monitoring is caused by the time-lapse-based embryo selection method itself or the uninterrupted culture environment that is part of the system. METHODS: In this three-armed, multicentre, double-blind, randomised controlled trial, couples undergoing in-vitro fertilisation or intracytoplasmic sperm injection were recruited from 15 fertility clinics in the Netherlands and randomly assigned using a web-based, computerised randomisation service to one of three groups. Couples and physicians were masked to treatment group, but embryologists and laboratory technicians could not be. The time-lapse early embryo viability assessment (EEVA; TLE) group received embryo selection based on the EEVA time-lapse selection method and uninterrupted culture. The time-lapse routine (TLR) group received routine embryo selection and uninterrupted culture. The control group received routine embryo selection and interrupted culture. The co-primary endpoints were the cumulative ongoing pregnancy rate within 12 months in all women and the ongoing pregnancy rate after fresh single embryo transfer in a good prognosis population. Analysis was by intention to treat. This trial is registered on the ICTRP Search Portal, NTR5423, and is closed to new participants. FINDINGS: 1731 couples were randomly assigned between June 15, 2017, and March 31, 2020 (577 to the TLE group, 579 to the TLR group, and 575 to the control group). The 12-month cumulative ongoing pregnancy rate did not differ significantly between the three groups: 50·8% (293 of 577) in the TLE group, 50·9% (295 of 579) in the TLR group, and 49·4% (284 of 575) in the control group (p=0·85). The ongoing pregnancy rates after fresh single embryo transfer in a good prognosis population were 38·2% (125 of 327) in the TLE group, 36·8% (119 of 323) in the TLR group, and 37·8% (123 of 325) in the control group (p=0·90). Ten serious adverse events were reported (five TLE, four TLR, and one in the control group), which were not related to study procedures. INTERPRETATION: Neither time-lapse-based embryo selection using the EEVA test nor uninterrupted culture conditions in a time-lapse incubator improved clinical outcomes compared with routine methods. Widespread application of time-lapse monitoring for fertility treatments with the promise of improved results should be questioned. FUNDING: Health Care Efficiency Research programme from Netherlands Organisation for Health Research and Development and Merck.

A prospective randomized comparison of sequential versus monoculture systems for in-vitro human blastocyst development.

BACKGROUND: Extending the period of in-vitro culture to the blastocyst stage may improve implantation rates in IVF treatment. Recognition of the dynamic nature of early embryo metabolism has led to the development of commercially available sequential culture systems. However, their improved efficacy over monoculture systems remains to be demonstrated in prospective studies. METHODS: Embryos obtained from 158 women undergoing IVF treatment were randomized by sealed envelopes to culture in one of three systems: (A) culture for 5 days in our own monoculture medium (Rotterdam medium); (B) culture for 3 days in Rotterdam medium followed by 2 days in fresh Rotterdam medium; (C) culture for 5 days using the commercially available G1/G2 sequential culture system. RESULTS: There were no significant differences in blastulation, implantation or pregnancy rates between the three tested culture systems. CONCLUSION: The employed monoculture system is as effective as the G1/G2 sequential system for the culture of blastocysts for IVF.

Genetic risk factors in infertile men with severe oligozoospermia and azoospermia.

BACKGROUND: Male infertility due to severe oligozoospermia and azoospermia has been associated with a number of genetic risk factors. METHODS: In this study 150 men from couples requesting ICSI were investigated for genetic abnormalities, such as constitutive chromosome abnormalities, microdeletions of the Y chromosome (AZF region) and mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. RESULTS: Genetic analysis identified 16/150 (10.6%) abnormal karyotypes, 8/150 (5.3%) AZFc deletions and 14/150 (9.3%) CFTR gene mutations. An abnormal karyotype was found both in men with oligozoospermia and azoospermia: 9 men had a sex-chromosomal aneuploidy, 6 translocations were identified and one marker chromosome was found. Y chromosomal microdeletions were mainly associated with male infertility, due to testicular insufficiency. All deletions identified comprised the AZFc region, containing the Deleted in Azoospermia (DAZ) gene. CFTR gene mutations were commonly seen in men with congenital absence of the vas deferens, but also in 16% of men with azoospermia without any apparent abnormality of the vas deferens. CONCLUSIONS: A genetic abnormality was identified in 36/150 (24%) men with extreme oligozoospermia and azoospermia. Application of ICSI in these couples can result in offspring with an enhanced risk of unbalanced chromosome complement, male infertility due to the transmission of a Y-chromosomal microdeletion, and cystic fibrosis if both partners are CFTR gene mutation carriers. Genetic testing and counselling is clearly indicated for these couples before ICSI is considered.

Implantation rates after in vitro fertilization and transfer of a maximum of two embryos that have undergone three to five days of culture.

OBJECTIVE: To evaluate implantation and pregnancy rates in patients undergoing IVF after the transfer of a maximum of two embryos that had been cultured for 3-5 days. DESIGN: Prospective study. SETTING: An IVF laboratory at a tertiary referral university hospital. PATIENT(S): One thousand seven hundred eighty-seven couples who underwent their first IVF cycle between January 1995 and December 1997. INTERVENTION(S): In vitro fertilization and transfer of embryos after 3, 4, or 5 days of culture using a single medium without coculture. MAIN OUTCOME MEASURE(S): Implantation and pregnancy rates. RESULT(S): Overall implantation and pregnancy rates were not significantly different with different culture periods. Forty-one percent of all available embryos developed into blastocysts on day 5. The transfer of at least one good-quality blastocyst could be performed in 62% of patients. Blastocysts had an implantation rate of 26% per embryo, whereas the implantation rate of eight-cell embryos on day 3 was 18%. Implantation rates for retarded, normal, and advanced embryos were not significantly different with an extended culture period. CONCLUSION(S): Under the study conditions, the transfer of embryos after 5 days rather than 3 days of embryo culture did not change the overall implantation and pregnancy rates. The implantation potential of embryos available for transfer can be assessed better after an extended culture period. Five days of culture allows the transfer of a reduced number of embryos without decreasing overall pregnancy rates.

Reproductive decisions of men with microdeletions of the Y chromosome: the role of genetic counselling.

Couples dealing with microdeletions of the Y chromosome have to make decisions about their reproductive future. Do they opt for intracytoplasmic sperm injection (ICSI), artificial insemination with donor insemination (AID) or no treatment? We analysed this decision in 28 couples and investigated the role of the counsellor and the counselling process on the final decision of the couple. Ten counsellors from six fertility clinics in The Netherlands and Belgium were interviewed about their genetic counselling of couples dealing with microdeletions. The answers to the questionnaire were converted to 11 dichotomous variables. Of the 1627 tested men in the six centres, 37 (2.3%) had a microdeletion in the AZFc region, a subregion of the AZF region on the Y chromosome important for normal spermatogenesis. The decisions of 28 of them could be analysed. Most couples chose ICSI (79%). The remaining couples chose donor insemination (7%) or refrained from treatment (14%). Several variables, including the counselling procedure, the counsellor and the available treatments in the fertility centre, influenced the decision of the couple. In conclusion, most couples dealing with microdeletions in the AZF region choose ICSI. Several aspects of the process of genetic counselling appear to be related to the final decision.

Subfertile men with constitutive chromosome abnormalities do not necessarily refrain from intracytoplasmic sperm injection treatment: a follow-up study on 75 Dutch patients.

A follow-up study was performed to investigate the impact of the detection of a chromosome abnormality in infertile men who are candidates for intracytoplasmic sperm injection (ICSI) treatment. In this collaborative study between clinical genetics centres and fertility clinics in the Netherlands, 75 ICSI couples of which the male partners had a chromosome abnormality were included. All couples were extensively counselled on the risk of having a chromosomally unbalanced child. Forty-two out of 75 couples chose to proceed with the ICSI treatment. So far, treatment has resulted in a pregnancy in 11 cases. Four of them opted to have invasive prenatal diagnosis. Despite the genetic risks related to a chromosome abnormality in infertile men, a small majority (56%) of the couples did not refrain from the ICSI treatment.

Retrieval, maturation, and fertilization of immature oocytes obtained from unstimulated patients with polycystic ovary syndrome.

PURPOSE: Our purpose was to determine whether immature oocytes could be retrieved under local anesthesia, whether these oocytes would mature and fertilize in vitro, and whether adequate endometrium development could be obtained after hormonal supplementation. METHODS: Ovum pick-up was performed under local anesthesia. Immature oocytes were cultured and inseminated. To prepare the endometrium, estradiolvalerate was administered in combination with micronized progesterone. RESULTS: Immature oocytes were obtained in all cases. Fifty-six percent (n = 30) of the oocytes developed into metaphase II (MII) after 48 hr of culture, and another 20% reached the MII stage by 72 hr. Normal fertilization was observed in only 10% of oocytes inseminated. No embryonic development occurred, and therefore embryo transfer was not performed in any of the patients. Endometrial microbiopsy was performed in all subjects and endometrial development was considered sufficient in eight patients. CONCLUSIONS: We collected immature oocytes from patients with polycystic ovary syndrome without general anesthesia. In vitro maturation of these oocytes seemed adequate but fertilization rates were poor. Sufficient endometrial quality was obtained after hormonal substitution.

Surgical sperm retrieval and intracytoplasmic sperm injection as treatment of obstructive azoospermia.

Male genital tract obstructions may result from infections, previous inguinal and scrotal surgery (vasectomy) and congenital bilateral absence of the vas deferens (CBAVD). Microsurgery can sometimes be successful in treating the obstruction. In other cases and in cases of failed surgical intervention, the patient can be treated by microsurgical or percutaneous epididymal sperm aspiration (MESA, PESA) or testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). We present the results of 39 ICSI procedures for obstructive azoospermia in 24 couples. The aetiology of the obstruction was failed microsurgery in 11 patients, CBAVD in nine and genital infections in four. Sperm retrieval was accomplished via MESA in four cases, PESA in 18 cases and via TESE in 11 cases. TESE was only applied when PESA failed to produce enough spermatozoa for simultaneous ICSI. In six patients, the ICSI procedure was performed with cryopreserved spermatozoa after an initial PESA procedure. Fertilization occurred in 47% of the metaphase II oocytes; embryo transfer was performed in 92% of procedures and resulted in a clinical pregnancy in 13/39 procedures. Ongoing pregnancy was achieved in 10/39 procedures. One pregnancy was terminated early after prenatal investigation showed a cytogenetic abnormality (47,XX+18, Edwards syndrome). The other nine pregnancies resulted in the live birth of 10 children, without any congenital abnormalities. Epididymal and testicular retrieved spermatozoa were successfully used for ICSI to treat obstructive azoospermia, and resulted in an ongoing pregnancy in 10 of 24 couples (41.6%) after 39 ICSI procedures, a success rate of 25.6% per treatment cycle and of 27.7% per embryo transfer.

Two cases of Robertsonian translocations in oligozoospermic males and their consequences for pregnancies induced by intracytoplasmic sperm injection.

Two case histories are presented documenting structural chromosome abnormalities in infertile males. The abnormalities were detected only after application of intracytoplasmic sperm injection (ICSI) was repeatedly unsuccessful or resulted in an abnormal pregnancy. A mosaic Robertsonian translocation 45,XY,der(13;13)(q10; q10)/46,XY,t(13;13)(p10;p10), der(13p;13p) incompatible with normal offspring was found in a male with extreme oligozoospermia after three subsequent ICSI treatments were unsuccessful and one had resulted in a spontaneous abortion. A second case involved a Robertsonian translocation 45,XY,der(13;14)(q10;q10) which was detected in a male with extreme oligozoospermia after ultrasound abnormalities were found in an ICSI-induced twin pregnancy. Amniocentesis showed an unbalanced 46,XY,+13,der(13;14)(q10;q10) karyotype in one twin and a Robertsonian 45,XX,der(13;14)(q10;q10) karyotype in the other twin. Chromosome analysis of males with abnormal sperm characteristics is advised prior to ICSI.

Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection.

Prenatal cytogenetic analysis of 71 fetuses conceived by intracytoplasmic sperm injection (ICSI) resulted in the detection of nine (12.7%) chromosome aberrations including two cases of 47,XXY, four cases involving a 45,X cell line and three autosomal trisomies. Molecular analysis of the parental origin of the deleted or supernumerary chromosome was performed by using polymorphic microsatellite markers. Six cases involving a sex chromosome abnormality were found to be of paternal origin while the two trisomic cases that could be analysed were of maternal origin. Two cases involved the same infertile couple who had two consecutive ICSI pregnancies terminated because of a chromosome abnormality. The replaced embryos in both cases originated from a single batch of ICSI fertilized oocytes of which part was used to initiate the first pregnancy and part was cryopreserved and used to initiate the second pregnancy.

Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa.

An infertile couple was referred for intracytoplasmic sperm injection (ICSI) because of primary infertility and oligoasthenoteratozoospermia (OAT) in the male. It was observed that although the sperm cells presented with an unusual head size and multiple tails they were able to fertilize the oocytes after ICSI. Subsequent molecular cytogenetic analysis demonstrated de-novo chromosome abnormalities in virtually all sperm cells with 40% diploidy and 24% triploidy in addition to aneuploidy for the sex chromosomes.

RS46(DXS548) genotyping of reproductive cells: approaching preimplantation testing of the fragile-X syndrome.

In order to approach preimplantation testing for the fragile-X syndrome, we used genotyping of the polymorphic RS46(DXS548) locus closely linked to the FMR-1 gene, in single reproductive cells of females. The RS46(DXS548) amplification was adjusted to the single cell level by a two-round polymerase chain reaction (PCR) procedure. Unfertilized oocytes and extruded polar bodies were subjected to PCR. RS46(DXS548) genotyping at the single cell level was successful in 95% of the samples. In two-third of the metaphase II oocytes and first polar bodies obtained from women who were heterozygous at the RS46(DXS548) locus, both maternal RS46(DXS548) alleles were observed because of crossing over during the first meiotic division. This makes gamete selection by first polar body analysis inefficient. From the allele frequencies found in 56 unrelated individuals, a heterozygote frequency of 51% was estimated, whereas the observed heterozygote frequency was 56%. The whole PCR procedure can be performed within 16 h after blastomere biopsy. Consequently, the selection and transfer of the diagnosed embryos can be carried out within an acceptable time. Therefore, preimplantation testing for the fragile-X syndrome with the RS46(DXS548) AC-repeat may be an alternative choice for prenatal testing for those carrier females who are heterozygous (informative) at the RS46(DXS548) locus.

Multiplex polymerase chain reaction for sex determination of single mouse blastomeres.

Using a multiplex nested polymerase chain reaction (PCR) method with single copy genes and a dinucleotide repeat locus for the mouse Y and X chromosomes respectively, it was possible to discriminate between single cells derived from male and female embryos. Using single cells, amplification of Sry and Zfy sequences was not evident in all cases. It could be calculated that, with the PCR method used, 0.04% [95% (confidence interval 0.00-2.03)] of the male embryos would erroneously be diagnosed as female if analysis is performed on two cells. The calculated chance for total amplification failure, if two cells are used for analysis, would be 1.4% [95% (confidence interval 0.04-8.04)]. The mouse embryo model proved to be a helpful tool to develop skills in the application of PCR for preimplantation genetic diagnosis at the single cell level.

Effect of oxygen concentration on in vitro fertilization and embryo culture in the human and the mouse.

OBJECTIVE: To compare the effect of culturing oocytes, zygotes and embryos under low (5%) versus ambient (20%) oxygen conditions on human IVF results and on mouse blastocyst formation. DESIGN: A prospective, randomized study of 257 consecutive IVF treatment cycles in 186 couples undergoing oocyte retrieval for various reasons of infertility. Gametes and resulting embryos after IVF were alternately allocated to fertilization and culture either under a gas phase of 5% CO2/90% N2/5% O2, or 5% CO2/95% air (20% O2). Oocytes and embryos from randomly bred and hybrid mouse strains were randomly allocated to culture under either of the two gas phases. SETTING: A university hospital-based IVF-ET program. MAIN OUTCOME MEASURE: In the human, rates of fertilization, embryonic development at the time of embryo replacement (42 to 46 hours after insemination), pregnancy, and implantation were compared. In the mouse, the rates of blastocyst formation were compared. RESULTS: Clinical pregnancies occurred in 24.2% versus 19.4% of retrievals when culture took place under low oxygen versus ambient oxygen conditions. Fertilization, embryonic development, pregnancy, and implantation rates did not differ significantly between the groups. Slightly higher blastocyst rates occurred when mouse embryos from hybrid strains were cultured under low oxygen compared with culture under ambient oxygen conditions, whereas no such difference in blastocyst rates was found in randomly bred mouse embryos. CONCLUSIONS: This study failed to demonstrate any improvement in human IVF results associated with the use of a gas mixture of 5% CO2/90% N2/5% O2 during the first two days of development compared with the use of 5% CO2 in air.

The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos.

OBJECTIVES: To evaluate the occurrence of injury due to physical factors in embryo cryopreservation and the effect of the polymers dextran, polyvinylpyrrolidone (PVP), and Ficoll on this mechanical damage. DESIGN: Damage to the zona pellucida (ZP) observed after cryopreservation was taken as indication of cryoinjury caused exclusively by physical factors. Human and mouse ZPs from oocytes remaining unfertilized after previous IVF attempts and mouse two-cell embryos were frozen in the presence of different polymers. After thawing, they were checked carefully for signs of physical damage (cracks). A possible toxicity of the use of the polymers in cryoprotection was evaluated by development to the blastocyst stage of mouse two-cell embryos that survived the freezing and thawing process. RESULTS: Incidences of damaged ZPs in groups of human and mouse ZPs and two-cell embryos frozen without polymers were found to vary between 20% and 29%. The use of any of the tested polymers resulted in significantly lower incidences of damaged ZPs (0% to 15%). Damage to the ZP after freezing and thawing in mouse embryos was accompanied by low survival rates of the embryo itself. Of mouse embryos that survived the cryopreservation process, blastocyst formation was not significantly different in groups frozen without polymer (80%) or in the presence of either dextran (90%) or Ficoll (82%); however, embryos frozen in the presence of PVP showed low blastocyst formation (12%). CONCLUSIONS: Polymers can protect embryos against cryoinjury by avoiding mechanical strain occurring during cryopreservation. Polyvinylpyrrolidine is toxic to mouse two-cell embryos when present during freezing and thawing.

Modulation of embryonic Na(+)-K(+)-ATPase activity and mouse preimplantation development in vitro in media containing high concentrations of potassium.

The effect of various potassium concentrations (ranging from 1.4 mM to 30 mM K+) in modified Tyrode's medium on the culture of mouse zygotes obtained after in vitro fertilization to the blastocyst stage was examined. A clear dose-dependent negative effect of increasing K+ concentrations on the preimplantation embryonic development in vitro was found. We have previously shown that significantly more two-cell embryos reach the blastocyst stage when cultured during the second day postinsemination in medium supplemented with taurine. Because taurine, an amino acid that abounds in the reproductive tract, has been reported to inhibit the enzyme Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase), we used two other conditions known to inhibit the Na(+)-K(+)-ATPase to study their effect on mouse embryo development. Culturing embryos during a short period (the second day postinsemination) in low extracellular K+ concentrations (1.4 mM) or in medium supplemented with ouabain (50 microM) showed positive effects similar to those of culturing in medium with taurine (10 mM). This beneficial effect of ouabain was found in various K+ concentrations tested, including the high concentrations present in the oviduct. Although the effects of low K+ and taurine can possibly be ascribed to their other cellular effects, the effect of ouabain shows that inhibition of the Na(+)-K(+)-ATPase during the two-cell stage in the mouse is beneficial for further embryonic development to the blastocyst stage.

Temporal effects of ouabain on in-vitro development of mouse zygotes.

Ouabain is a specific inhibitor of Na(+)-K(+)-ATPase, an enzyme which controls the intracellular Na+ and K+ levels. In this study, in-vitro fertilized zygotes from a hybrid mouse strain were used to examine the temporal effects of 50 microM ouabain on embryonic development in vitro during the preimplantation period. A higher incidence of blastocyst formation at the end of the culture period was found when embryos were cultured in the presence of ouabain from 22 to 46 h post-insemination, or any other period that included this time period. When zygotes from randomly bred mice were used, inhibition of Na(+)-K(+)-ATPase with ouabain clearly promoted development through the 2-cell block in vitro. As Na(+)-K(+)-ATPase is the most important regulator of intracellular electrolyte concentrations in mammalian cells, these results suggest that an ionic imbalance exists in embryos cultured in conventional media which can be positively influenced by inhibiting this enzyme.

Detection of aneuploidy and chromosomal mosaicism in human embryos during preimplantation sex determination by fluorescent in situ hybridisation, (FISH).

Five couples at risk of producing offspring with X-linked recessive disease underwent in vitro fertilisation with a view to preimplantation determination of embryo sex and selective transfer of females. On day three postinsemination, one or two blastomeres were removed by embryo biopsy, and used for dual fluorescent in situ hybridisation with X and Y chromosome-specific DNA probes. In two cases, two female embryos were transferred and one pregnancy, (sex confirmed), is ongoing at 19 weeks. All eight embryos from one couple were of such poor quality that diagnosis was possible in one only. In the remaining two cases no embryos were transferred due to the detection of an abnormal number of X chromosome signals. Investigation of the biopsied embryos that were not transferred revealed evidence of mitotic non-disjunction in one and of complete X monosomy in a second. A surviving fetus with this latter constitution would have developed Turner syndrome and would also have been at high risk of X-linked disease. The use of fluorescent in situ hybridisation rather than the polymerase chain reaction allowed the detection of abnormal copy numbers of X chromosomes thus preventing the transfer of potentially abnormal zygotes.

Predictive value of morphologically normal sperm concentration in the medium for in-vitro fertilization.

Morphological evaluation of spermatozoa using strict criteria (MEUSC) and conventional sperm parameters were studied with respect to in-vitro fertilization and pregnancy outcome before and after a swim-up selection procedure. Recovered oocytes were inseminated with 50,000 progressively motile spermatozoa, and this study assess the influence of the total number of spermatozoa and of the percentage with strictly normal morphology in the insemination sample on the outcome of IVF. The results showed that the percentages of spermatozoa with normal morphology using strict criteria, both in native and in post-swim-up samples, were the best predictors of IVF outcome. Their respective cut-off points were 5% and 8%. The number of morphologically normal spermatozoa inseminated also showed a good correlation with fertilization. However, it was not possible to find a proper cut-off point for this parameter. The patients were categorized on the basis of their native and post-swim-up scores. Category 1, in which both parameters were below their respective cut-off points, showed a 7% fertilization rate and a 0% pregnancy rate. Category 3, in which both parameters were above their cut-off points, showed a 70% fertilization rate and a 23% pregnancy rate. This suggests that sperm morphology can be used as a criterion for patient selection for IVF as an aid to identification of possibly subfertile males.