In assisted reproductive treatments, embryos remaining after fresh embryo transfer are usually selected for cryopreservation based on traditional morphology assessment. Our previous report has demonstrated that array comparative genomic hybridization (aCGH) screening for IVF patients with good prognosis significantly improves clinical and ongoing pregnancy rates in fresh embryo transfer cycles. The current study further investigates the efficiency of applying aCGH in the selection of euploid embryos for cryopreservation as related to pregnancy and implantation outcomes in subsequent frozen embryo transfer (FET) cycles.
First-time IVF patients with good prognosis undergoing fresh single embryo transfer and having at least one remaining blastocyst for cryopreservation were prospectively randomized into two groups: 1) Group A patients had embryos assessed by morphology first and then by aCGH screening of trophectoderm cells and 2) Group B patients had embryos evaluated by morphology alone. All patients had at least one blastocyst available for cryopreservation after fresh embryo transfer. There were 15 patients in Group A and 23 patients in Group B who failed to conceive after fresh embryo transfer and completed the FET cycles. Blastocyst survival and implantation rates were compared between the two groups.
There were no significant differences in blastocyst survival rates between Group A and Group B (90.9% vs. 91.3%, respectively; p >0.05). However, a significantly higher implantation rate was observed in the morphology assessment plus aCGH screening group compared to the morphology assessment alone group (65.0% vs. 33.3%, respectively; p = 0.038). There was no miscarriage observed in Group A while a 16.7% miscarriage rate was recorded in Group B (0% vs. 16.7%, respectively; p >0.05).
While aCGH screening has been recently applied to select euploid blastocysts for fresh transfer in young, low-risk IVF patients, this is the first prospective study on the impact of aCGH specifically on blastocyst survival and implantation outcomes in the subsequent FET cycles of IVF patients with good prognosis. The present study demonstrates that aCGH screening of blastocysts prior to cryopreservation significantly improves implantation rates and may reduce the risk of miscarriage in subsequent FET cycles. Further randomized clinical studies with a larger sample size are needed to validate these preliminary findings.
aCGH; Trophectoderm biopsy; Cryopreservation; Implantation
Background: IVF pregnancy rates have trended upward although gains have been accompanied by unwelcome increases in pre-term delivery and multiple gestation. These adverse outcomes happen because multiple embryos are typically transferred during IVF. Integrating newer molecular cytogenetic techniques with IVF can optimize selection of a single embryo for transfer. Methods: The SurePlex DNA amplification system (BlueGnome Ltd; Cambridge, UK) was used on-site for whole genome amplification of human blastocyst trophectoderm (TE) cells obtained by biopsy. IVF patients (initial cycle, age <35, no prior miscarriage, normal karyotype) were prospectively randomized into two groups: In Group 1, embryos were selected on the basis of morphology and comprehensive chromosomal screening via array comparative genomic hybridization (aCGH) from d5 TE biopsy, while Group 2 embryos were assessed by morphology only. All patients underwent a single fresh blastocyst transfer on d6. For embryos in the aCGH group, only one euploid blastocyst was selected for transfer and surplus euploid blastocysts were vitrified. In the non-aCGH (control) group, a single blastocyst was selected for fresh transfer based on appearance only, with vitrification of any surplus blastocysts with satisfactory morphology. Results: Aneuploidy was identified in 191/425 of Group 1 balstocysts (44.9%). Control embryos (n=389) were assessed by microscopy only. A higher clinical pregnancy rate was observed in Group 1 patients compared to the control group (70.9 vs. 45.8%; p = 0.017). Only 64 (28.3%) surplus euploid embryos were frozen in Group 1 while 157 (40.4%) blastocysts were cryopreserved for Group 2 (p=0.017). Conclusion: These data underscore the intrinsic imprecision of IVF when conventional morphology is used alone to select embryos for transfer. Embryos evaluated with aCGH implant with greater efficiency and achieve clinical pregnancy more often than those selected without aCGH. Patients should be advised that aCGH screening may reduce the number of surplus embryos for cryopreservation.
During IVF, non-transferred embryos are usually selected for cryopreservation on the basis of morphological criteria. This investigation evaluated an application for array comparative genomic hybridization (aCGH) in assessment of surplus embryos prior to cryopreservation.
First-time IVF patients undergoing elective single embryo transfer and having at least one extra non-transferred embryo suitable for cryopreservation were offered enrollment in the study. Patients were randomized into two groups: Patients in group A (n=55) had embryos assessed first by morphology and then by aCGH, performed on cells obtained from trophectoderm biopsy on post-fertilization day 5. Only euploid embryos were designated for cryopreservation. Patients in group B (n=48) had embryos assessed by morphology alone, with only good morphology embryos considered suitable for cryopreservation.
Among biopsied embryos in group A (n=425), euploidy was confirmed in 226 (53.1%). After fresh single embryo transfer, 64 (28.3%) surplus euploid embryos were cryopreserved for 51 patients (92.7%). In group B, 389 good morphology blastocysts were identified and a single top quality blastocyst was selected for fresh transfer. All group B patients (48/48) had at least one blastocyst remaining for cryopreservation. A total of 157 (40.4%) blastocysts were frozen in this group, a significantly larger proportion than was cryopreserved in group A (p=0.017, by chi-squared analysis).
While aCGH and subsequent frozen embryo transfer are currently used to screen embryos, this is the first investigation to quantify the impact of aCGH specifically on embryo cryopreservation. Incorporation of aCGH screening significantly reduced the total number of cryopreserved blastocysts compared to when suitability for freezing was determined by morphology only. IVF patients should be counseled that the benefits of aCGH screening will likely come at the cost of sharply limiting the number of surplus embryos available for cryopreservation.
Fertilization in vitro; Comparative genomic hybridization; Preimplantation genetic diagnosis; Cryopreservation
The objective of our study was to determine if trophectoderm biopsy, vitrification, array-comparative genomic hybridization and single thawed euploid embryo transfer (STEET) can reduce multiple gestations and yield high pregnancy and low miscarriage rates.
We performed a retrospective observational study comparing single thawed euploid embryo to routine age matched in vitro fertilization (IVF) patients that underwent blastocyst transfer from 2008 to 2011 and to our best prognosis group donor oocyte recipients (Donor). Our main outcome measures were implantation rate, clinical pregnancy rate, spontaneous abortion rate and multiple gestation rate.
The STEET group had a significantly higher implantation rate (58 %, 53/91) than the routine IVF group (39 %, 237/613) while the Donor group (57 %, 387/684) had a similar implantation rate. The clinical pregnancy rates were not statistically different between the STEET and IVF groups. However, the multiple gestation rate was significantly lower in the STEET group (STEET 2 % versus IVF 34 %, Donor 47 %).
STEET results in a high pregnancy rate, low multiple gestation rate and miscarriage rates. Despite the older age of STEET patients and transfer of twice as many embryos, the implantation rate for STEET was indistinguishable from that for egg donation. STEET offers an improvement to IVF, lowering risks without compromising pregnancy rate.
Trophectoderm biopsy; Single embryo transfer; Array-comparative genomic hybridization; Aneuploidy; Embryo biopsy; Donor egg; IVF; Embryo transfer
Single embryo transfer (SET) provides the most certain means to reduce the risk of multiple gestation. Regrettably, prospective trials of SET have demonstrated reductions in per-cycle delivery rates. A validated method of comprehensive chromosome screening (CCS) has the potential to optimize SET by transferring only euploid embryos. This retrospective study evaluates the efficacy of SET with CCS in an infertile population.
Overall and age-controlled ongoing pregnancy rates (OPR) were compared between women undergoing SET following CCS (CCS-SET, n= 140) and those undergoing SET without aneuploidy screening (control SET, n= 182). All transfers were at the blastocyst stage, with CCS performed after trophectoderm biopsy of expanded blastocysts and analysis with rapid PCR allowing for fresh transfer.
In the CCS-SET and control SET groups, an OPR of 55.0 and 41.8%, respectively, was obtained. The OPR was lower for the control group (P< 0.01) despite a younger age than the CCS group (37.3 ± 3.4 versus 34.2 ± 3.9 years; P< 0.001). Birthweight and gestational age at delivery were equivalent. The proportion of clinical pregnancies resulting in miscarriage was higher in the control group (24.8 versus 10.5%, P< 0.01), with more patients requiring surgical interventions for aneuploid pregnancies. There was one monozygotic twin delivery in the CCS group and none in the control group.
Compared with traditional blastocyst SET, SET after trophectoderm biopsy and rapid PCR-based CCS increases OPR and reduces the miscarriage rate. The enhanced selection empowered by CCS with SET may provide a practical way to eliminate multi-zygotic multiple gestation without compromising clinical outcomes per cycle.
single embryo transfer; IVF; comprehensive chromosome screening; multiple gestation; pregnancy rate
Chromosomal abnormalities are common in embryos produced in vitro and cause implantation failure, miscarriage, and serious medical problems in infants. Because preimplantation genetic screening (PGS) is increasingly being used to detect aneuploidy in embryos with the purpose of improving implantation rates after IVF (in vitro fertilization), we aimed to validate the usefulness of array CGH for the preimplantation genetic screening (PGS) of embryos at the blastocyst stage of development.
A total of 150 blastocysts were biopsied from couples undergoing IVF and analyzed using array CGH. We found that 54.5% (73/134) of the blastocysts were euploid embryos, whereas 45.5% of the embryos (61/134) had chromosomal abnormalities. Multiple chromosome abnormality was most frequently observed (34.4%), and dual aneuploidy was observed in 26.2% of the embryos. Monosomy (21.3%) appeared more frequently than trisomy (18%).
Chromosomal microarray analysis provided clinically significant cytogenetic information regarding the frequency and variety of chromosomal abnormalities observed in embryos at the blastocyst stage, suggesting that this is a useful tool for comprehensive aneuploidy screening in IVF.
Single blastocyst transfer has the advantage of maximizing the fresh single pregnancy rate. However, in patients with a low number of good quality embryos on day 3, it remains unclear whether immediate embryo transfer or further embryo culture with blastocyst transfer is the most preferable option.
A retrospective cohort study was carried out in which the outcome of 590 fresh in vitro fertilization (IVF) cycles over a 15 months period and their cryo cycles were analyzed. A total of 341 patients cycles had an elective day 5 strategy independent of intermediate embryo evaluation while another 249 patients underwent a day 5 embryo transfer only if at least four embryos were available on day 3. Blastocyst vitrification was performed using a closed high security system.
Demographics, stimulation parameters and embryological data were comparable in the two groups. Patients in the elective day 5 group had a lower fresh transfer rate (90.62% vs. 95.18%, p < 0.05) as compared to patients with a day 3 or day 5 embryo transfer policy. No difference was observed in the fresh live birth rate and multiple pregnancy rate per initiated cycle (32.84% vs. 28.92%; 1.17% vs 0%) The projected cumulative ongoing pregnancy rate compensating for double counting in case subjects have more than one pregnancy is not different (42.58% vs. 39.84%).
Despite lower fresh transfer rates, elective single blastocyst transfer yields a similar projected cumulative ongoing pregnancy rate as in a policy with cleavage stage or blastocyst transfer depending on a good quality embryo count on day 3.
Despite an ongoing debate over its efficacy, preimplantation genetic screening (PGS) is increasingly being used to detect numerical chromosomal abnormalities in embryos to improve implantation rates after IVF. The main indications for the use of PGS in IVF treatments include advanced maternal age, repeated implantation failure, and recurrent pregnancy loss. The success of PGS is highly dependent on technical competence, embryo culture quality, and the presence of mosaicism in preimplantation embryos. Today, cleavage stage biopsy is the most commonly used method for screening preimplantation embryos for aneuploidy. However, blastocyst biopsy is rapidly becoming the more preferred method due to a decreased likelihood of mosaicism and an increase in the amount of DNA available for testing. Instead of using 9 to 12 chromosome FISH, a 24 chromosome detection by aCGH or SNP microarray will be used. Thus, it is advised that before attempting to perform PGS and expecting any benefit, extended embryo culture towards day 5/6 should be established and proven and the clinical staff should demonstrate competence with routine competency assessments. A properly designed randomized control trial is needed to test the potential benefits of these new developments.
Preimplantation genetic screening; Aneuploidy; In vitro fertilization; Embryo quality; Embryo biopsy
We present a series of monozygous multiple gestations achieved following in vitro fertilization (IVF): one case of monochorionic triplet pregnancy and six cases of dizygotic triplet pregnancy. From September 2000 to December 2006, all patients achieving clinical pregnancy by ART were reviewed (n = 2433). A 37 year-old woman who delivered a healthy singleton after IVF returned two years later for FET, and a single blastocyst was transferred. This also resulted in pregnancy, but TV-USG revealed a single gestational sac with three distinct amniotic sacs, each containing a distinct fetal pole with cardiac activity. This pregnancy was electively terminated at nine weeks' gestation. An additional six cases of dizygotic triplets established after fresh embryo transfer (no ICSI or assisted hatching) are also described. Of these, one resulted in a miscarriage at eight weeks' gestation and five patients have an ongoing pregnancy. This case series suggests the incidence of dizygotic/monochorionic triplets following IVF is approximately 10 times higher than the expected rate in unassisted conceptions, and underscores the importance of a conservative approach to lower the number of embryos at transfer. The role of embryo transfer technique and in vitro culture media in the twinning process requires further study.
Although selection of chromosomally normal embryos has the potential to improve outcomes for patients undergoing IVF, the clinical impact of aneuploidy screening by fluorescence in situ hybridization (FISH) has been controversial. There are many putative explanations including sampling error due to mosaicism, negative impact of biopsy, a lack of comprehensive chromosome screening, the possibility of embryo self-correction and poor predictive value of the technology itself. Direct analysis of the negative predictive value of FISH-based aneuploidy screening for an embryo's reproductive potential has not been performed. Although previous studies have found that cleavage-stage FISH is poorly predictive of aneuploidy in morphologically normal blastocysts, putative explanations have not been investigated. The present study used a single nucleotide polymorphism (SNP) microarray-based 24 chromosome aneuploidy screening technology to re-evaluate morphologically normal blastocysts that were diagnosed as aneuploid by FISH at the cleavage stage. Mosaicism and preferential segregation of aneuploidy to the trophectoderm (TE) were evaluated by characterization of multiple sections of the blastocyst. SNP microarray technology also provided the first opportunity to evaluate self-correction mechanisms involving extrusion or duplication of aneuploid chromosomes resulting in uniparental disomy (UPD). Of all blastocysts evaluated (n = 50), 58% were euploid in all sections despite an aneuploid FISH result. Aneuploid blastocysts displayed no evidence of preferential segregation of abnormalities to the TE. In addition, extrusion or duplication of aneuploid chromosomes resulting in UPD did not occur. These findings support the conclusion that cleavage-stage FISH technology is poorly predictive of aneuploidy in morphologically normal blastocysts.
FISH; PGD; microarray; mosaicism; uniparental isodisomy
In-vitro fertilization (IVF) with blastocyst as opposed to cleavage stage embryos has been advocated to improve success rates. Limited information exists on which to predict which patients undergoing blastocyst embryo transfer (BET) will achieve pregnancy. This study's objective was to evaluate the predictive value of patient and cycle characteristics for clinical pregnancy following fresh BET.
This was a retrospective cohort study from 2003–2007 at an academic assisted reproductive program. 114 women with infertility underwent fresh IVF with embryo transfer. We studied patients undergoing transfer of embryos at the blastocyst stage of development. Our main outcome of interest was clinical pregnancy. Clinical pregnancy and its associations with patient characteristics (age, body mass index, FSH, ethnicity) and cycle parameters (thickness of endometrial stripe, number eggs, available cleaving embryos, number blastocysts available, transferred, and cryopreserved, and embryo quality) were examined using Student's T test and Mann-Whitney-U tests as appropriate. Multivariable logistic regression models were created to determine independent predictors of CP following BET. Receiver Operating Characteristic analyses were used to determine the optimal thickness of endometrial stripe for predicting clinical pregnancy.
Patients achieving clinical pregnancy demonstrated a thicker endometrial stripe and were younger preceding embryo transfer. On multivariable logistic regression analyses, Caucasian ethnicity (OR 2.641, 95% CI 1.054–6.617), thickness of endometrial stripe, (OR 1.185, 95% CI 1.006–1.396) and age (OR 0.879, 95% CI 0.789–0.980) predicted clinical pregnancy. By receiver operating characteristic analysis, endometrial stripe ≥ 9.4 mm demonstrated a sensitivity of 83% for predicting clinical pregnancy following BET.
In a cohort of patients undergoing fresh BET, thicker endometrial stripe, Caucasian ethnicity, and younger age are positive predictors of clinical pregnancy after fresh BET. These findings may be useful in clinical management of infertile patients undergoing fresh BET cycles.
The aim of this study was to investigate the effect of chromosomal polymorphic variations on the outcome of IVF and embryo transfer (IVF–embryo transfer) treatment for infertile couples.
During the period from October 2006 to December 2009, 1978 infertile couples who had received their first IVF–embryo transfer treatment cycle in our hospital were selected for this retrospective study, and the frequency of chromosomal polymorphic variations was calculated. From these, 1671 couples were selected and divided into three groups: 1402 couples with normal chromosomes (Group 1/control group), 82 couples with chromosomal polymorphic variations in only females (Group 2) and 187 couples with chromosomal polymorphic variations in only males (Group 3). The clinical pregnancy rates (CPR), early miscarriage rates and ongoing pregnancy rates after IVF–embryo transfer treatment were compared.
There were no statistically significant differences among the three groups in implantation rates (29.37% in the control group, 29.70% in Group 2 and 31.41% in Group 3, P > 0.05) and CPR (45.86, 46.34 and 51.87%, respectively, P > 0.05). Although there was a trend toward higher first trimester pregnancy loss rates in Group 3 (male chromosomal polymorphic variations), but not in Group 2, compared with normal karyotype couples (10.31 versus 6.84%), the difference did not reach significance (P > 0.05).
Chromosomal polymorphic variations appear to have no adverse effects on the outcome of IVF–embryo transfer treatment.
chromosome polymorphism; IVF; pregnancy rate; early miscarriage
In IVF-ICSI cycles with single embryo transfer (SET), embryo selection for transfer is of crucial importance. The present study aimed to define which embryo parameters might be related to the implantation potential of advanced blastocysts.
Overall, in 203 cycles with SET, developmental characteristics of 93 implanted (group A) and 110 non-implanted (group B) advanced blastocysts of good quality were compared. The following developmental parameters were assessed in the two groups: normal fertilization, developmental stage on day 5, number of blastomeres on day 2 and on day 3, fragmentation rate on day 3, compaction on day 4 and cleavage pattern on day 2 and day 3.
Expanded blastocysts compared to full blastocysts have higher implantation potential (56.5% vs. 29.3%, p < 0.05). In group B, a higher proportion of advanced blastocysts showed between 10% and 50% anucleated fragments on day 3 than in group A (23.6 vs 11.8, P = 0.03). Advanced blastocysts with >10–50% fragments on day 3 showed a significant lower implantation (29.7%) than those with ≤ 10%fragments (49.4%, P = 0.03). All the other parameters analysed were comparable for the two groups.
Developmental stage on day 5 and fragmentation rate on day 3 were related to the implantation potential of advanced blastocysts and should also be taken into account in the selection of the best advanced blastocyst for transfer.
To investigate the effect of long zona dissection (LZD) compared with partial zona dissection (PZD) using ICSI pipettes for mechanical assisted hatching (AH) in vitrified-thawed blastocyst transfers.
University IVF clinic.
A total of 120 women ≦ 38 years old undergone vitrified-thawed blastocyst transfers with LZD or PZD.
The culture of all pronucleate embryos to the blastocyst stage and the selection of blastocysts ≧ grade 3BB (Gardner and Schoolcraft score), followed by vitrified-thawed blastocyst transfers with LZD (n = 60) or with PZD (n = 60)
Main outcome measure(s)
Complete hatching rates, implantation rates, pregnancy rates.
At 5 h after thawing, complete hatching rates of blastocysts were significantly higher in LZD group compared with PZD group, 52.4 % vs. 31.8 % (P = 0.001). Implantation and clinical pregnancy rates were significantly higher in LZD group compared with PZD group, 40.9 % vs. 25.7 % and 63.0 % vs. 40.0 %, respectively (P = 0.010, P = 0.011).
LZD using ICSI pipettes for mechanical AH improves significantly complete hatching, implantation and pregnancy rates in vitrified-thawed blastocyst transfers.
Long zona dissection; ICSI pipettes; Assisted hatching; Vitrified-thawed blastocyst transfers
To examine the clinical pregnancy rate (CPR) and multiple pregnancy rate (MPR) in a large in vitro fertilisation (IVF) programme before and after the introduction of single blastocyst transfer (SBT) strategy in a selected group of women.
A 3-year pre- and postintervention study.
A tertiary reproductive medicine and assisted conception unit in a London teaching hospital.
Two thousand four hundred and fifty-one fresh IVF cycles performed between July 2004 and June 2007 at the Assisted Conception Unit at Guy’s and St Thomas’ Hospital NHS Foundation Trust were included in the study.
In January 2006, we implemented a multidisciplinary intervention involving the introduction of a selective day 5 SBT service together with an educational programme on the risks of multiple pregnancy and potential advantages of blastocyst transfer aimed at couples at high risk of multiple pregnancy.
Main outcome measures
The CPR per cycle started and MPR per clinical pregnancy achieved.
A statistically significant increase in the CPR from 27% (324/1198) to 32% (395/1253) (risk difference [RD] 5%, risk ratio [RR] 1.17, 95% CI 1.03–1.32, P = 0.015) and reduction in the MPR per clinical pregnancy from 32% (103/272) to 17% (69/395) (RD 15%, RR 0.46, 95% CI 0.35–0.60, P < 0.001) were observed after introduction of the SBT service.
Selective SBT in women with good prognosis can reduce the MPR after IVF while maintaining the overall success rate of the IVF programme.
Please cite this paper as:Khalaf Y, El-Toukhy T, Coomarasamy A, Kamal A, Bolton V, Braude P. Selective single blastocyst transfer reduces the multiple pregnancy rate and increases pregnancy rates: a pre- and postintervention study. BJOG 2008;115:385–390.
Blastocyst transfer; clinical pregnancy; IVF; multiple pregnancy; single embryo transfer pregnancy
Management of repeated implantation failure despite transfer of good-quality embryos still remains a dilemma for ART specialists. Scrapping of endometrium in the nontransfer cycle has been shown to improve the pregnancy rate in the subsequent IVF/ET cycle in recent studies.
The objective of this randomized controlled trial (RCT) was to determine whether endometrial injury caused by Pipelle sampling in the nontransfer cycle could improve the probability of pregnancy in the subsequent IVF cycle in patients who had previous failed IVF outcome.
Tertiary assisted conception center.
Randomized controlled study.
MATERIALS AND METHODS:
100 eligible patients with previous failed IVF despite transfer of good-quality embryos were randomly allocated to the intervention group and control groups. In the intervention group, Pipelle endometrial sampling was done twice: One in the follicular phase and again in the luteal phase in the cycle preceding the embryo transfer cycle.
The primary outcome measure was live birth rate. The secondary outcome measures were implantation and clinical pregnancy rates.
The live birth rate was significantly higher in the intervention group compared to control group (22.4% and 9.8% P = 0.04). The clinical pregnancy rate in the intervention group was 32.7%, while that in the control group was 13.7%, which was also statistically significant (P = 0.01). The implantation rate was significantly higher in the intervention group as compared to controls (13.07% vs 7.1% P = 0.04).
Endometrial injury in nontransfer cycle improves the live birth rate, clinical pregnancy and implantation rates in the subsequent IVF-ET cycle in patients with previous unsuccessful IVF cycles.
Endometrial injury; IVF/ICSI; Pipelle biopsy
To compare the clinical outcomes between fresh and vitrified-thawed day 5 blastocyst transfers.
Retrospective case control study.
Tertiary referral center.
Patients 38 years of age or less who underwent IVF/ICSI cycles with fresh or frozen-thawed blastocysts transferred from June 1, 2009 to November 30, 2011
Vitrification and thawing of day 5 blastocysts using the Cryotop method. (Kitazato BioPharma Co., Ltd., Fuji city, Shizuoka, Japan)
Main outcome measure(s)
Clinical pregnancy rate, implantation rate, ongoing pregnancy rate, and multiple pregnancy rates.
Of the 118 cycles in the fresh transfer group, 234 blastocysts were transferred. The clinical pregnancy rate was 66.1 % and implantation rate was 50.9 %. The ongoing pregnancy rate was 56.8 % and the rates for singleton and twin pregnancies were 53.7 % and 44.8 %. Of the 59 cycles in the vitrified-thawed group, 111 blastocysts were transferred. The clinical pregnancy rate was 59.3 % and implantation rate was 43.2 %. The ongoing pregnancy rate was 47.5 % and the rates for singleton and twin pregnancies were 60.7 % and 39.3 %. The clinical pregnancy rate, implantation rate and ongoing pregnancy rate did not differ significantly between the two groups.
The implantation rates were not significantly different between the fresh and the vitrified-thawed groups. Thus, single embryo transfer may be considered in fresh cycles to decrease multiple pregnancy rates. The surplus embryos should be vitrified for the frozen embryo transfer to improve the cumulative pregnancy rate.
Vitrification; Blastocyst; Implantation; Pregnancy
Most in vitro fertilization (IVF) programs employ embryo cryopreservation to enhance pregnancies from a single ovarian stimulation. More embryos are created, some of which are not transferred to the uterus immediately, generating a need for improved cryopreservation protocols. One protocol may involve growing embryos to a further stage of development, allowing only embryos with proven developmental capabilities to be cryopreserved. Here we examined thaw survival, implantation and live birth rates of embryos cryopreserved at different stages.
We examined thaw survival, implantation and live birth rates of embryos cryopreserved at the zygote, day 3 (D3) embryos or blastocyst stage.
SETTINGS AND DESIGN:
This is a retrospective study from a single academic IVF program.
PATIENTS AND METHODS:
A retrospective study of all patients who had frozen embryos transferred to their uteri from year 2002 to 2008 at a single academic IVF program was conducted.
STATISTICAL ANALYSIS USED:
Analysis of variance followed by Fisher's Exact Test was performed to compare the survival after thaw, implantation and live birth rates between the three groups.
One thousand nine hundred and ninety-one zygotes, 2880 D3 embryos and 503 blastocysts were frozen using a slow freeze technique, thawed and transferred. Significantly more D3 embryos and blastocysts survived the thawing process compared to zygotes and significantly higher implantation rate per number of thawed blastocysts was achieved than that for zygotes. Live birth rates were similar between the three groups.
Growing embryos to blastocyst stage prior to cryopreservation is associated with fewer frozen embryos but does not appear compromise patients’ chance of achieving pregnancy
Blastocyst; embryo cryopreservation; frozen embryo transfer; implantation; in vitro fertilization; slow-freeze; zygote
Metabolomics was introduced in human in vitro fertilization (IVF) for noninvasive identification of viable embryos with the highest developmental competence.
To determine whether embryo selection using a commercial version of metabolomic analysis leads to increased implantation rates (IRs) with fetal cardiac activity (FCA) compared with morphology evaluation alone.
SETTING AND DESIGN:
Randomized controlled trial from April to December 2010 at a private IVF unit. The study was terminated prematurely due to the market withdrawal of the instrument.
MATERIALS AND METHODS:
IVF patients ≥18 and ≤43 years with ≥4 × 2PN were randomly allocated to metabolomic analysis combined with embryo morphology (ViaMetrics-E; metabolomics + morphology group) or embryo morphology alone (morphology group). Cycles with frozen embryos, oocyte donations, or testicular biopsy were excluded.
Categorical and continuous data were analyzed for statistical significance using 2-tailed Fisher's exact test and t-test, respectively. Statistical significance was accepted when P > 0.05.
A total of 125 patients were included in the study; 39 patients were allocated to metabolomics + morphology group and 86 patients to morphology group. Patients were stratified according to the day of embryo transfer (Days 2, 3, or 5). IRs with FCA were similar for Days 2 and 3 transfers in both groups. For Day 5 transfers, IRs with FCA were significantly higher in the metabolomics + morphology group (46.8% vs. 28.9%; P = 0.041; 95% confidence intervalp [CI]: 1.09-34.18). Pregnancy and live births rates were similar for Days 2, 3, and 5 in both groups. The study was terminated early following the voluntary market withdrawal of ViaMetrics-E in December 2010.
Metabolomic analysis using the commercial near-infrared (NIR) instrument does not appear to have a beneficial effect on pregnancy and live births, with improvement in IR with FCA for Day 5 transfers. However, no solid conclusions can be reached due to the lack of adequate study power.ClinicalTrials.gov Identifier: NCT01490515
Embryo selection; implantation rates; metabolomics; near-infrared; noninvasive
Purpose: To investigate the relative power of HCG, estradiol, and progesterone determinations in the prediction of pregnancy outcome after IVF. These prognostic hormonal factors were studied as single and combined predictors.
Methods: Serum concentrations of β-HCG, progesterone, and estradiol were measured 12–13 days after embryo transfer (study point 1) and 7 days later (study point 2) in a series of 20 consecutive infertile patients having a first-trimester spontaneous clinical abortion after an IVF-embryo transfer cycle. As a control group (n=60), the next three IVF-embryo transfer cycles resulting in an ongoing pregnancy after each miscarried IVF cycle in our assisted reproduction program was used. The discrimination attained between the two study groups (ongoing pregnancies and miscarriages) was evaluated by logistic regression and receiver operating characteristic (ROC) curve analysis.
Results: Mean hormone concentrations at study points 1 and 2 were higher in the ongoing pregnancy than in the abortion group. Regarding pregnancy outcome the percentage increment of HCG serum levels (≥1321%), with an accuracy (predictive value of pregnancy outcome) of 81.2% (sensitivity 98%, specificity 50%), had the best prognostic reliability but no significant differences were found when this parameter was compared with the predictive value of HCG concentration (≥72 IU/l) at study point 1 (diagnostic accuracy 80.5%; sensitivity 70%; specificity 80%). When ROC analysis was used, the best predictor of ongoing pregnancy according to the AUCROC was HCG concentration at study point 2 but again no significant differences were found when this parameter was compared with the predictive value of HCG serum levels at study point 1. A multiple marker strategy did not help distinguish viable from nonviable pregnancies.
Conclusion: A single, early (days 12–13 after embryo transfer) HCG quantitative serum measurement in IVF cycles not only is diagnostic but also has good predictive value for pregnancy outcome.
Abortion; HCG; IVF; pregnancy outcome; progesterone
One of the most important factors influencing embryo viability is chromosome imbalance (aneuploidy). Embryos derived from aneuploid gametes have little potential for forming a viable pregnancy, but cannot be distinguished from normal embryos using standard morphological evaluation. For more than a decade, preimplantation genetic screening (PGS) has been used to assist in the identification of aneuploid embryos. However, current strategies, based upon cell biopsy followed by fluorescent in situhybridization, allow less than half of the chromosomes to be screened. In this review, we discuss methods that overcome the limitations of earlier PGS strategies and provide screening of the entire chromosome complement in oocytes and embryos. In recent months, there has been a rapid growth in the number of PGS cycles utilizing one such method, comparative genomic hybridization (CGH). Data from IVF cycles utilizing CGH must be considered preliminary, but appear to indicate a dramatic increase in embryo implantation following comprehensive chromosomal screening. It is expected that methods based upon microarrays will yield similar clinical results and may be sufficiently rapid to permit comprehensive screening without the need for embryo cryopreservation. Some microarray platforms also offer the advantage of embryo fingerprinting and the potential for combined aneuploidy and single gene disorder diagnosis. However, more data concerning accuracy and further reductions in the price of tests will be necessary before microarrays can be widely applied.
comparative genomic hybridization; aneuploidy; preimplantation genetic screening; in vitro fertilization; preimplantation genetic diagnosis
Purpose: To investigate whether ICSI (intracytoplasmic sperm injection)results in decreased blastocyst formation and pregnancy compared to IVF (in vitro fertilization).
Methods: We performed a retrospective analysis of blastocyst transfer (BT)offered routinely to patients under age 40 with ≥ three 8-cell embryos on day 3 and compared IVF to ICSI cycles. Sequential media were used with P1 until day 3, then Blastocyst Medium until day 5/6.
Results: There were 131 IVF and 75 ICSI cycles. There was no difference in age, number of oocytes, zygotes, 8-cell embryos, blastocysts on days 5 and 6, or embryos transferred. Progression to blastocyst was similar (78% for IVF and 73% for ICSI) as was the viable pregnancy rate (51.4% for IVF and 55% for ICSI). No cycles failed to form blastocysts.
Conclusions: The progression to blastocyst and the likelihood of conceiving aviable pregnancy were unaltered by ICSI. Thus it seems appropriate for programs to offer BT to patients undergoing ICSI using the same inclusion criteria applied to their IVF patients.
Blastocyst; ICSI; IVF; male factor infertility; pregnancy rate
High proportions of human embryos produced by in vitro fertilization are aneuploidy and mosaic. DNA microarray is one of the most practical screening methods to select euploid embryos for transfer. However, mosaic pregnancy is still possible due to embryonic mosacism. Here we report a successful pregnancy after transfer of a mosaic blastocyst with euploid inner cell mass.
A woman with a previous trisomy 13 pregnancy pursued infertility treatment with preimplantation genetic screening by a trophectoderm biopsy and DNA microarray. NimbleGen oligonucleotide DNA microarray was applied to biopsied samples from 13 blastocysts. A euploid blastocyst was transferred to the patient and subsequent prenatal cytogenetic tests were performed by FISH and/or G banding.
Following DNA microarray, it was found that 5 blastocysts were euploid and 8 were aneuploidy. Transfer of one euploid blastocyst resulted in a clinical pregnancy. Prenatal cytogenetic tests of samples biopsied from chorionic villi sample showed both trisomy 21 (47 XX, +21) and euploid (46, XX) cells. Further prenatal cytogenetic test with a sample from amniotic fluid indicated that all cells were euploid (46, XX). The pregnancy was continued and a healthy girl was delivered after 41 weeks of gestation.
This is the first report to indicate a mosaic pregnancy after transfer of a “euploid” blastocyst that was screened by DNA microarray, and the case further confirms that mosaicism is present in human blastocysts produced by in vitro fertilization.
Mosaicism; Blastocyst; Pregnancy; Human
Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.
Purpose: The aim of this prospective, randomized trial was to evaluate the efficacy of Embryo-Glue® as a human embryo transfer medium in IVF/ICSI cycles.
Method: A total of 815 nonselected patients undergoing IVF/ICSI treatment between September 2003 and February 2004 were randomly allocated into the test (417 patients) and the control (398 patients) groups. In both groups, embryos were cultured in G-1™ver 3, supplemented with 10% recombinant human albumin. On the day of embryo transfer (day 3), the best or good quality embryos were selected for intrauterine transfer. In the test group, the selected embryos were treated with EmbryoGlue® prior to the transfer, whereas in the control group they were transferred without any treatment.
Results: The patients’ characteristics such as age and the number of ART cycles and also the number of patients in each indication of infertility and the number of embryos selected for transfer were all similar between the two groups. In the test group, the clinical pregnancy rate in the tubal factors and the implantation rate in the tubal factors and recurrent implantation failures increased significantly compared with those in the control group. In the test group, life birth and the triplet delivery rates increased significantly compared with those in the control group.
Conclusion: EmbryoGlue® is a useful embryo transfer medium, and at least in some infertile patients it can improve clinical implantation and ongoing pregnancy rates.
Clinical pregnancy rate; Embryoglue®; implantation rate; recurrent implantation failures; Tubal factors