Analysis of 122 triplet and one quadruplet pregnancies after single embryo transfer in Japan.

RESEARCH QUESTION: What is the prevalence of triplet and quadruplet pregnancies after single embryo transfer (SET) in Japan. DESIGN: A retrospective observational study was conducted on 274,605 pregnancies after 937,848 SET cycles in registered assisted reproductive technology (ART) data from the Japanese ART national registry database between 2007 and 2014. A questionnaire survey of ART centres was also conducted. Data on pregnancies with embryo division into three or more after SET were analysed. RESULTS: According to the Japanese ART national registry database, SET resulted in 109 triplet pregnancies (0.04% of pregnancies), and the questionnaire reports from 31 centres revealed 33 triplet and one quadruplet pregnancies. After exclusion of 20 duplicated cases, 122 triplet and one quadruplet pregnancies included 46 monochorionic (one gestational sac [37.4%]), 18 dichorionic (two gestational sacs [14.6%]) and 59 trichorionic pregnancies (three gestational sacs [48.0%]). Compared with singleton pregnancies, patients with monozygotic triplet or quadruplet pregnancies were less frequently diagnosed with unexplained infertility (P = 0.004), more often received gonadotrophin injections for ovarian stimulation in 39 cases with information available (P = 0.021) and underwent more blastocyst transfers and assisted hatching (P = 0.002 and P < 0.001, respectively). The proportion of live birth, defined as at least one baby born, excluding induced abortion, was 64.6% (73/116 pregnancies) of monozygotic triplet or quadruplet pregnancies. CONCLUSIONS: Combined Japanese ART national registry and survey data revealed 122 triplet and one quadruplet pregnancies, the majority after cryopreserved embryo transfer. Most were conceived after blastocyst transfer and often after assisted hatching, which are potential risk factors for zygotic splitting.

Endogenous progesterone levels could predict reproductive outcome in frozen embryo replacement cycles supplemented with synthetic progestogens: A retrospective cohort study.

PURPOSE: A retrospective, cohort study was conducted between 2009 and 2017 in a private infertility center to determine the predictive value of endogenous estrogen (E2) and progesterone (P4) levels in hormone-replacement frozen embryo replacement (FER) treatment cycles. METHODS: A total of 120 consecutive, infertile patients who became pregnant after FER cycles were analyzed (age: 37.4 ± 4.4 years). Electively vitrified blastocysts were created during natural cycle IVF or mild ovarian stimulation treatments and subsequently transferred through delayed vitrified-thawed blastocyst transfer cycles supplemented with estrogens and a combination of synthetic progestogens. Serum E2 and progesterone P4 levels were intensively monitored every five days (from the day after embryo transfer until 9w1d of pregnancy) and compared among patients with a subsequent live birth (n = 76) or first-trimester pregnancy loss (n = 44). RESULTS: Endogenous placental activity started as early as 5-6th pregnancy week differing significantly according to pregnancy outcome. For P4, the exponential rise from 6w2d onwards allowed distinguishing between failing and successful conceptions. For P4, lower quartiles of the live birth group did not intersect with upper quartiles of the miscarriage group. CONCLUSIONS: Innovative FER protocols incorporating synthetic progestogens allow the correct measurement of endogenous placental activity and could help to monitor early first-trimester ART pregnancies.

Predicting live birth by combining cleavage and blastocyst-stage time-lapse variables using a hierarchical and a data mining-based statistical model.

Prolonged embryo culture is increasingly used as a way of improving pregnancy rates, especially in the context of single embryo transfer. So far, only a handful of studies examined the relation between implantation potential and time-lapse parameters extracted from later stages (morula and blastocyst) of embryo development. For this retrospective study all 285 single vitrified-thawed blastocyst transfers (SVBT) from all consecutive unselected patients whose fertilized oocytes were submitted to time-lapse monitoring (TLM) from a two-year cohort were analysed. Two different statistical models were created; a hierarchical one including the two strongest live birth (LB) predictors (t2 and texpB2) and a more complex model based on principal component analysis (PCA) and logistic regression methods. The first, four-category, hierarchical model effectively distinguished between blastocysts of increasing LB rates (8, 30, 40, 53%). For the second data-mining model quartiles of the created Sc parameter had increasing LB rates (12, 19, 40, 49%). AUC values were comparable for both models (0.723, 95CI%:0.66-0.79 versus 0.717, 95CI%:0.65-0.78). The combination of cleavage- and blastocyst-stage variables through hierarchical or data mining-based algorithms was used successfully to predict live birth. However, due to the lack of internal / external validation the predictive capacities of this model could differ largely in different datasets.

Blastocyst collapse is not an independent predictor of reduced live birth: a time-lapse study.

OBJECTIVE: To ascertain the rate of blastocyst collapse observed by time-lapse monitoring in a retrospective cohort of unselected infertile patients undergoing single blastocyst transfer and to determine its association with live birth. DESIGN: Blastocyst collapse and morphokinetic variables were scored according to previously published criteria. The association between blastocyst collapse and live birth was evaluated by a multivariate logistic regression analysis including morphokinetic variables and other confounders. SETTING: Private infertility clinic. PATIENT(S): Patients who underwent 277 consecutive single blastocyst transfers (mean age, 38.4 ± 3.9 years; range, 28-47 years) after minimal ovarian stimulation. INTERVENTION(S): Minimal ovarian stimulation, prolonged embryo culture in time-lapse monitoring incubator, elective vitrification with subsequent vitrified-warmed single blastocyst transfer. MAIN OUTCOME MEASURE(S): Live birth rate per single blastocyst transfer in different blastocyst collapse groups (no, single, multiple collapses). RESULT(S): No, single, or multiple blastocyst collapses occurred in 54% (150/277), 22% (61/277), and 24% (66/277) of the cohort, respectively. In the multiple collapse group on average 2.9 contractions were seen (range, 2-9 contractions). Live birth rate decreased progressively between blastocyst collapse groups (36%, 31%, 14%); significantly lower if multiple collapses occurred. In a multivariate analysis, however, blastocyst collapse was not found to be a significant predictor and was confounded by stronger predictors such as morphokinetic variables t2, texpB2, and female age. CONCLUSION(S): Blastocyst collapse pattern should not be evaluated alone without taking into account morphokinetic variables that are stronger predictors of reproductive outcome.

Time-lapse variables and embryo gender: a retrospective analysis of 81 live births obtained following minimal stimulation and single embryo transfer.

PURPOSE: The purpose of this study was to determine which morphokinetic variables are related to embryo gender in a cohort of consecutive live births obtained through single blastocyst transfer following mild ovarian stimulation. METHODS: Eighty-one live births (49 % of them females) from successfully treated, consecutive infertile patients (maternal age 36.9 ± 3.8 years, range 28-46) who underwent minimal ovarian stimulation, prolonged embryo culture in a time-lapse monitoring (TLM) incubator and elective single blastocyst transfers during 2012-2014. Early (PNf, t2-t9, cc2a, b, s2, s3) and late (tM, tSB, tfullB, texpB1, and texpB2) morphokinetic variables were scored according to published consensus criteria and were normalized to the time of pronuclear fading. For each variable, the ranges with the highest proportion of female embryos (optimal range) were determined by detailed examination of histograms. RESULTS: Female embryo gender was associated both with late cleavage (t8), morula (tM), and blastocyst stage morphokinetic variables. The strongest associations (adjusted ORs, 7.0-7.8) were found for late, expanded stage blastocyst parameters; tfullB, texpB1, and texpB2. The proportion of female embryos was 69-71 and 25-26 % inside and outside of the optimal ranges, respectively. This allowed to predict 74-78 % of them, increasing their proportion by 57 % compared to the average. CONCLUSIONS: Although the sample size of our cohort was limited, our findings suggest that several expanded blastocyst stage morphokinetic parameters are associated with female embryo gender. If confirmed on a larger sample these could be potentially used to increase the proportion of female embryos among non-invasively selected blastocysts following single embryo transfer.

Influence of different oocyte insemination techniques on early and late morphokinetic parameters: retrospective analysis of 500 time-lapse monitored blastocysts.

OBJECTIVE: To determine how standard IVF vs. intracytoplasmic sperm injection (ICSI) fertilization influences early and late morphokinetic parameters during prolonged embryo culture. DESIGN: Five-hundred expanded blastocysts that were monitored in a time-lapse monitoring incubator were analysed retrospectively. Early (pronuclear fading [PNf], t2-t9) and late (start of blastulation, expanded blastocyst) morphokinetic variables were scored according to published consensus criteria. SETTING: Private infertility clinic. PATIENT(S): A total of 209 consecutive infertile patients (mean ± SD age, 38.4 ± 4 years; range, 28-47 years) undergoing 238 natural IVF/minimal ovarian stimulation cycles during 2012-2014. INTERVENTION(S): Minimal ovarian stimulation, oocyte retrieval, fertilization with standard IVF or ICSI, prolonged embryo culture in a time-lapse monitoring incubator. MAIN OUTCOME MEASURE(S): Differences in morphokinetic parameters according to insemination techniques. RESULT(S): In total, 29% and 71% of the whole cohort was fertilized with standard IVF and ICSI, respectively. During early cleavage stages (PNf to t4) there was a statistically significant delay (+1.5 to +1.1 hours) among IVF-fertilized embryos. By contrast, at the expanded blastocyst stage IVF-fertilized embryos showed faster development (-3.3 to -4.1 hours). After normalizing to the time point of PNf, differences in cleavage-stage parameters disappeared, but those at all blastocyst stages increased even further in favor of IVF-fertilized embryos (-3.2 to -5.7 hours). CONCLUSION(S): The observed 1.5-hour time difference between standard IVF- and ICSI-fertilized embryos is an artificial phenomenon. At the blastocyst stages, however, genuine timing differences arise between IVF- and ICSI-fertilized embryos, possibly related to their different quality. Normalization to a common time point permits the joint analysis of IVF- and ICSI-fertilized embryos, thus increasing the size of studied cohorts.

Time-lapse monitoring of zona pellucida-free embryos obtained through in vitro fertilization: a retrospective case series.

OBJECTIVE: To report time-lapse monitoring of human oocytes in which the damaged zona pellucida was removed, producing zona-free (ZF) oocytes that were cultured until the blastocyst stage in time-lapse incubators. DESIGN: Retrospective case series. SETTING: Private infertility clinic. PATIENT(S): Infertile patients (n = 32) undergoing minimal ovarian stimulation or natural cycle IVF treatment between October 2012 and June 2014. INTERVENTION(S): Intracytoplasmic sperm injection (ICSI) fertilization of ZF oocytes, prolonged embryo culture in time-lapse incubators, elective vitrification, and subsequent single vitrified-thawed blastocyst transfer (SVBT). MAIN OUTCOME MEASURE(S): Rate of fertilization, cleavage and blastocyst development, live-birth rate per SVBT cycle. RESULT(S): In spite of advanced maternal age (39 ± 4.2; range, 30-46 years), good fertilization (94%), cleavage (94%), and blastocyst development rates (38%) were reached after fertilization and culturing of ZF oocytes/embryos. All thawed ZF blastocysts survived, and up to this date seven SVBT transfers were performed, yielding three (43%) term live births with healthy newborns. CONCLUSION(S): Time-lapse imagery gives a unique insight into the dynamics of embryo development in ZF embryos. Moreover, our case series demonstrate that an oocyte with a damaged zona pellucida that has been removed could be successfully fertilized with ICSI, cultured until blastocyst stage in a time-lapse incubator and vitrified electively for subsequent use.

Cumulative success rates following mild IVF in unselected infertile patients: a 3-year, single-centre cohort study.

A 3-year, retrospective, single-centre cohort study was conducted in a private infertility centre to determine cumulative live birth rates (LBR) per scheduled oocyte retrieval following minimal ovarian stimulation/natural-cycle IVF in unselected infertile patients. A total of 727 consecutive infertile patients were analysed who underwent 2876 (median 4) cycles with scheduled oocyte retrieval from November 2008 to December 2011. Natural-cycle IVF or clomiphene-based minimal ovarian stimulation was coupled with single-embryo transfer and increased use of delayed vitrified-warmed blastocyst transfer. Main outcome measures were crude and expected age-specific cumulative LBR per scheduled oocyte retrieval. Crude cumulative LBR were 65%, 60%, 39%, 15% and 5% in patients aged 26-34, 35-37, 38-40, 41-42 and 43-44 years, respectively. No live births occurred in patients aged ⩾ 45 years. Dropout rates per cycle were 13-25%. Success rates gradually reached a plateau, with few additional live births after six cycles. Most of the expected success rate was reached within 6 months with almost maximal rates within 15 months of the first oocyte retrieval. Acceptable cumulative LBR are reached with an exclusive minimal ovarian stimulation/single-embryo transfer policy especially in patients aged <38 years but also in intermediate aged patients (38-40 years).

Oocyte retrieval timing based on spontaneous luteinizing hormone surge during natural cycle in vitro fertilization treatment.

OBJECTIVE: To determine the efficiency of oocyte retrieval (OR) timing based on the occurrence of spontaneous LH surge during natural cycle IVF (ncIVF) treatment. DESIGN: Retrospective cohort study. The cohort was divided into five subgroups according to the presumed stage of spontaneous LH surge on scheduling day (1A: before onset; 1B: surge start; 2: ascending slope; 3: peak; and 4: descending slope). SETTING: Private infertility clinic. PATIENT(S): Three hundred sixty-five infertile patients who underwent 1,138 ncIVF treatment cycles during 2008-2011. INTERVENTION(S): Drug-free ncIVF treatment. MAIN OUTCOME MEASURE(S): Rate of successfully retrieved, fertilized oocytes, cleaved embryos, and live births per scheduled oocyte retrieval. RESULT(S): In 61% of the cycles OR was scheduled before or just at the start of the LH surge (groups 1A-1B), whereas in the remaining cases it was scheduled after the surge had already started (groups 2-4). The proportion of cycles with successfully recovered (range, 71%-86%), inseminated (range, 61%-78%), fertilized oocytes (range, 47%-68%), cleaved embryos (range, 45%-66%), and live births (range, 4.1%-9.2%) was not significantly different among subgroups. CONCLUSION(S): In ncIVF treatment OR timing based on the occurrence of spontaneous LH surge is feasible, yielding acceptable oocyte recovery, fertilization, and embryo cleavage rates. This strategy combined with a rapid and low-risk OR procedure permits the management of a large ncIVF program on a 7-days-per-week basis within working hours.

Minimal ovarian stimulation combined with elective single embryo transfer policy: age-specific results of a large, single-centre, Japanese cohort.

BACKGROUND: The two main complications associated with the use of assisted reproduction techniques, ovarian hyperstimulation syndrome and multiple pregnancies, could be eliminated by milder ovarian stimulation protocols and the increased use of a single embryo transfer (SET) policy. A retrospective, cohort study was performed in private infertility centre to evaluate the embryological and clinical results of a large exclusively SET program according to patient age (lower or equal 29, 30-34, 35-39, 40-44 and equal or higher 45 years). MATERIALS: A total of 7,244 infertile patients have undergone 20,244 cycles with a clomiphene-based minimal stimulation or natural cycle IVF protocol during 2008. Following oocyte retrieval, fertilization and embryo culture a total of 10,401 fresh or frozen single embryo transfer procedures were performed involving cleavage-stage embryos or blastocysts. RESULTS: Successful oocyte retrieval rate (78.0 %) showed no age-dependent decrease until 45 years. Fertilization (80.3 %) and cleavage (91.1 %) rates were not significantly different between age groups. Blastocyst formation (70.1 % to 22.8 %) and overall live birth rates (35.9 % to 2 %) showed an age-dependent decrease. Frozen-thawed blastocyst transfer cycles gave the highest chance of live birth per embryo transfer (41.3 % to 6.1 %). CONCLUSIONS: High fertilization and cleavage rates were obtained regardless of age whereas blastocyst formation and live birth rates showed an age-dependent decrease. An elective single embryo transfer program based on a minimal ovarian stimulation protocol yields acceptable live birth rates per embryo transfer in infertile patients up until their mid-forties. However in very advanced age patients (equal or higher 45 years old) success rates fall below 1 %.

Short-term, low-dose, non-steroidal anti-inflammatory drug application diminishes premature ovulation in natural-cycle IVF.

A retrospective cohort study was conducted in a private infertility centre to evaluate the use of non-steroidal antiinflammatory drugs (NSAID) in natural-cycle IVF (nIVF) treatment. A total of 1865 first-rank nIVF cycles performed during 2009­2010 were evaluated. Low-dose, post-trigger NSAID was administered in a non-randomized way in cycles at higher ovulation risk where an imminent LH surge was detected on triggering day. Main outcome measures were premature ovulation rate, embryo transfer rate per scheduled cycle and clinical pregnancy and live birth rates per embryo transfer. NSAID use was associated with a significantly lower risk of premature ovulation (3.6% versus 6.8%, adjusted OR 0.24, 95% CI 0.15­0.39, P < 0.0001) and higher embryo transfer rate (46.8% versus 39.5%, adjusted OR 1.38, 95% CI 1.06­1.61, P = 0.012) per scheduled cycle. Clinical pregnancy (39.1% versus 35.9%) and live birth rates per embryo transfer (31.3% versus 31.4%) were comparable. In this retrospective series, short-term low-dose NSAID application positively influenced nIVF cycles by diminishing the rate of unwanted premature ovulations and increasing the proportion of cycles reaching embryo transfer.

Neonatal outcome and birth defects in 6623 singletons born following minimal ovarian stimulation and vitrified versus fresh single embryo transfer.

OBJECTIVE: To compare neonatal outcome between children born after vitrified versus fresh single-embryo transfer (SET). STUDY DESIGN: Retrospective, single-centre cohort study of 6623 delivered singletons following 29,944 single-embryo transfers. Patients underwent minimal ovarian stimulation/natural cycle IVF followed by SET of fresh or vitrified-warmed (using Cryotop, Kitazato) cleavage-stage embryos or blastocysts. Outcome measures were gestational age at delivery, birth weight, birth length, low birth weight (LBW), small for gestational age (SGA) and large for gestational age (LGA) infants, perinatal mortality and minor/major birth defects (evaluated by parent questionnaire). RESULTS: Gestational age (38.6 ± 2 versus 38.7 ± 1.9 weeks) and preterm delivery rate (6.9% versus 6.9%, aOR: 0.96 95%CI: 0.76-1.22) in singletons born after the transfer of vitrified embryos were comparable to those born after the transfer of fresh embryos. Children born after the transfer of vitrified embryos had a higher birth weight (3028 ± 465 versus 2943 ± 470 g, p<0.0001) and lower LBW (8.5% versus 11.9%, aOR: 0.65 95%CI: 0.53-0.79) and SGA (3.6% versus 7.6% aOR: 0.43 95%CI: 0.33-0.56) rates. Total birth defect rates (including minor anomalies) (2.4% versus 1.9%, aOR: 1.41 95%CI: 0.96-2.10) and perinatal mortality rates (0.6% versus 0.5%, aOR: 1.02 95%CI: 0.21-4.85) were comparable between the vitrified and fresh groups. CONCLUSIONS: Vitrification of embryos/blastocysts did not increase the incidence of adverse neonatal outcomes or birth defects following single embryo transfer.

Blastocyst culture is associated with an elevated incidence of monozygotic twinning after single embryo transfer.

In a 7-year (2002-2008) retrospective study of a large IVF program based on minimal ovarian stimulation and single ET (47,841 single ETs), monozygotic twinning occurred in 1.01% of 14,956 clinical pregnancies. Blastocyst culture was associated with a significantly increased monozygotic twinning risk (adjusted odds ratio, 2.04; 95% confidence interval, 1.29-4.48), whereas embryo freezing, type of stimulation protocol used, intracytoplasmic sperm injection fertilization, or zona removal did not influence its incidence.

Successful pregnancy, achieved by ovulation induction using a human menopausal gonadotropin low-dose step-up protocol in an infertile patient with Kallmann's syndrome.

A 25-year-old woman, diagnosed with Kallmann's syndrome and wanting to become pregnant, visited our hospital. Because her serum gonadotropin levels indicated hypogonadotropic hypogonadism, a main symptom of Kallmann's syndrome, we attempted to induce ovulation using a low-dose human menopausal gonadotropin (hMG) step-up protocol. In this protocol, 75 IU of hMG was used as an initial dose and this was continued for the first 14 days because adequate follicular development was not achieved. The dose of hMG was subsequently increased to 150 IU for the next 7 days. After 22 days from the start of stimulation, two follicles had developed, and were ovulated using an injection of human chorionic gonadotropin. She became pregnant, and her pregnancy was uneventful during the first trimester; however, in the second trimester both uterine contractions and blood pressure could not be controlled, and at 27 weeks' gestation she delivered a male infant weighing 830 g by cesarean section.

Eight-hydroxy-2'-deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program.

OBJECTIVE: To determine the effects of oxidative stress on the quality of oocytes and embryos, 8-hydroxy-2'-deoxyguanosine (8-OHdG) in granulosa cells was quantitatively studied during an in vitro fertilization and embryo transfer (IVF-ET) program. DESIGN: Immunocytochemical staining of 8-OHdG in granulosa cells was quantitatively estimated using a charge-coupled device camera and analyzed using the National Institute of Health Image (NIH Image) freeware on a computer . SETTING: Obstetrics and gynecology department in a university hospital. PATIENT(S): Ninety-six infertile couples undergoing IVF-ET treatment and intracytoplasmic sperm injection (IVF, n = 72; intracytoplasmic sperm injection, n = 24). INTERVENTION(S): Oocytes, granulosa cells, and follicular fluids were collected 35-36 hours after the administration of hCG. MAIN OUTCOME MEASURE(S): 8-OHdG indices were obtained for mural [8-OHdG index (m)] and cumulus [8-OHdG index (c)] granulosa cells. RESULT(S): A negative correlation between the fertilization rate and both 8-OHdG indices (c and m) was found. The rate of production of good embryos also showed a negative correlation with the 8-OHdG index (m) and the 8-OHdG index (c). Negative correlations between the 8-OHdG index (c) and E2 levels in follicular fluid were observed. Endometriosis patients showed a higher 8-OHdG index (c) than did patients with other infertility causes, such as tubal, male factor, and unknown. CONCLUSION(S): Oxidative stress in granulosa cells lowered fertilization rates and subsequently led to a decrease in the quality of embryos. The quality of oocytes for endometriosis patients was impaired by the presence of 8-OHdG. This might be one causative factor in infertility in endometriosis patients.