Successful birth following transfer of frozen–thawed embryos produced from in-vitro matured oocytes

It is well established that ovarian hyperstimulation syndrome (OHSS) is more frequent in patients with polycystic ovarian syndrome. In-vitro maturation (IVM) of immature oocytes presents a potential alternative for the fertility treatment and prevention of OHSS for these patients. This report describes the case of a 26-year old woman with a successful pregnancy and delivery following the transfer of frozen–thawed embryos derived from in-vitro matured oocytes. She had three failed cycles of ovarian stimulation (using low-dose step-up gonadotrophin protocol) with or without intrauterine insemination cycles, an ovulation-induction cycle with luteal long protocol, two fresh IVM cycle and one frozen–thawed IVM cycle. During the IVF cycle, she developed moderate OHSS and required hospitalization for 3 weeks. Following four unsuccessful IVF or IVM cycles, 15 months after the last cryopreservation, six fertilized oocytes were thawed for a scheduled embryo transfer. Following thawing, four fertilized oocytes survived and cleaved. Four frozen–thawed embryos were transferred. Six weeks after embryo transfer an ongoing intrauterine single pregnancy with fetal heartbeat was confirmed by transvaginal ultrasound. An uneventful pregnancy and delivery via Caesarean section at 39 weeks resulted in the birth of a normal healthy infant.     

Live birth from vitrified–warmed human oocytes fertilized with frozen–thawed testicular spermatozoa

A couple with male infertility due to non-obstructive azoospermia were referred to the fertility centre for treatment. Testicular biopsy was performed on the male partner and testicular samples were frozen. The female partner underwent ovarian stimulation and 31 mature oocytes were recovered by ultrasound-guided vaginal aspiration. Twelve oocytes were cryopreserved by the Cryotop vitrification method and 19 oocytes were inseminated by intracytoplasmic sperm injection (ICSI) using frozen–thawed testicular spermatozoa. Nine out of 19 oocytes were fertilized and the resulting embryos were cryopreserved by slow freezing. Four months later, two out of six thawed embryos were transferred, but no pregnancy resulted. One year later, the couple decided to attempt pregnancy using vitrified oocytes and frozen testicular spermatozoa. Six vitrified–warmed oocytes were injected with frozen–thawed testicular spermatozoa and four were fertilized. On the day of transfer, two cleavage stage embryos (4-cell, 2-cell) were obtained. Serum β-HCG test 14 days after embryo transfer was positive. Hormonal support for the established pregnancy was maintained with oestradiol and progesterone orally until 12 weeks of gestation. A healthy baby boy weighing 3.09 kg was delivered by elective Caesarean section at 38 weeks of gestation. This case report demonstrates that oocyte cryopreservation by the Cryotop vitrification method does not compromise oocyte developmental competence.     

HCG administration after endogenous LH rise negatively influences pregnancy rate in modified natural cycle for frozen–thawed euploid blastocyst transfer: a pilot study

Purpose

The aim of the present study was to evaluate whether in a modified natural cycle (modified-NC) for a frozen-thawed single euploid blastocyst transfer, a critical LH value, above which human chorionic gonadotropin (hCG) administration should be avoided, may be defined.

Methods

One hundred and sixty-seven patients underwent modified natural cycle in order to transfer a single frozen-thawed euploid blastocyst. All embryos were obtained by intracytoplasmic sperm injection and were biopsied at the blastocyst stage and analyzed by means of array comparative genomic hybridization (aCGH). Ovulation was induced using 10.000 IU hCG when the mean follicle diameter was at least of 17 mm, independently from LH values. The primary end points were the hCG-positive test and clinical pregnancy. The interim analysis showed that LH value ≥?13 mIU/ml on the day of hCG injection may negatively influence the clinical results, suggesting that in this condition, it should be advisable waiting for spontaneous ovulation.

Results

Among patients who received hCG for ovulation induction, the hCG-positive test and clinical pregnancy rates in modified-NC were significantly lower in cycles with LH?≥?13 mIU/ml in respect to those with LH?<?13 mIU/ml (45.4 vs 73.3 and 36.4 vs 65.9%, in LH?≥?13 and LH?<?13 groups, respectively). In patients with LH value ≥?13 mIU/ml, hCG administration led to significantly lower rates of hCG-positive test (45.4 vs 74.5% in hCG administration and spontaneous ovulation groups, respectively) and clinical pregnancy (36.4 vs 64.7% in hCG administration and spontaneous ovulation groups, respectively). The baseline patient characteristics were comparable in all groups.

Conclusions

The findings of this study highlight that LH elevation ≥?13 mIU/ml prior to hCG administration may negatively affect clinical pregnancy rates in modified-NC for single euploid blastocyst transfer. The LH determination should be routinely performed during follicular monitoring. In the presence of LH level ≥?13 mIU/ml, hCG administration should be avoided, and the embryo transfer should be planned only after spontaneous follicular rupture.

     

What is the preferred method for timing natural cycle frozen–thawed embryo transfer?

Spontaneous ovulation during a natural menstrual cycle represents a simple and efficient method for synchronization between frozen embryos and the endometrium. The objective was to compare serial monitoring until documentation of ovulation, with human chorionic gonadotrophin (HCG) triggering, for timing frozen embryo transfer (FET) in natural cycles (NC). In a retrospective study, 112 women with regular menstrual cycles undergoing 132 NC–FET cycles were divided into two groups: group A (n = 61) patients had FET in an NC after ovulation triggering with HCG; group B (n = 71) patients had FET in an NC after spontaneous ovulation was detected. The main outcome measure was the number of monitoring visits at the clinic. Patients in both groups were similar in terms of demographic characteristics and reproductive history. Clinical and laboratory characteristics of fresh and frozen cycles were also found comparable for both groups, as were pregnancy and delivery rates. The number of monitoring visits in group A (3.46 ± 1.8) was significantly lower than in group B (4.35 ± 1.4) (P < 0.0001). In patients undergoing NC–FET, triggering ovulation by HCG can significantly reduce the number of visits necessary for cycle monitoring without an adverse effect on cycle outcome. Ovulation triggering can increase both patient convenience and cycle cost-effectiveness.     

Predictive value of serum kisspeptin concentration at 14 and 21 days after frozen–thawed embryo transfer

Research questionCan serum kisspeptin levels 14 and 21 days after frozen–thawed embryo transfer predict the early pregnancy outcome of patients?DesignProspective study, with 133 patients undergoing frozen–thawed embryo transfer. Patients were divided into non-pregnant group and pregnant group (including biochemical pregnancy, singleton pregnancy, miscarriage and twin groups).ResultsSerum kisspeptin levels on day 21 were significantly higher than day 14 in singleton pregnancy, miscarriage and twin groups (all P < 0.0001), but not in the biochemical pregnancy group. Similarly, serum human chorionic gonadotrophin (HCG) levels were higher on day 21 compared with day 14 except for the biochemical pregnancy group. Compared with the twin group (296.9 pg/ml), the other four groups showed significantly higher serum kisspeptin levels on day 14 (non-pregnant 548.9, biochemical pregnancy 440.4, miscarriage 434.9, singleton pregnancy group 420.9 pg/ml, P < 0.01, P = 0.016, P = 0.034, P = 0.036, respectively). The miscarriage (762.2 pg/ml), singleton pregnancy (730.8 pg/ml) and twin groups (826.3 pg/ml) had significantly higher kisspeptin levels than the biochemical pregnancy group (397.3 pg/ml) on day 21 (P < 0.001, P < 0.01, P < 0.001, respectively). Serum kisspeptin levels on day 14 were negatively correlated with embryo implantation rate (P = 0.035, R² = –0.880). Serum kisspeptin levels on day 21 have a poor predictive value of miscarriage compared with serum HCG levels (area under the curve = 0.53 and 0.78, respectively).ConclusionsSerum kisspeptin levels on day 14 are negatively correlated with embryo implantation rate. Serum kisspeptin levels on day 21 have a poor predictive value of miscarriage.     

Letrozole ovulation induction: an effective option in endometrial preparation for frozen–thawed embryo transfer

Purpose

To evaluate the clinical efficacy of letrozole on ovulation induction and hormone replacement therapy (HRT) during endometrial preparation for frozen–thawed embryo transfer (FET).

Methods

We analyzed totally 1,230 cycles of patients that underwent FET from October 2010 to September 2012. Seven hundred and thirteen cycles of patients with ovulation disorders that underwent FET were randomly assigned to two groups by case control study. 359 cycles received letrozole ovulation induction and 354 cycles received HRT during endometrial preparation for FET, respectively. In the corresponding period, 517 cycles of patients with normal ovulation in the natural cycle group for FET endometrial preparation served as controls. Reproduction-related clinical outcomes of patients in the three groups were compared.

Results

The embryo implantation rate of patients in letrozole group (30.4 %) was significantly higher than the HRT group (22.8 %, P < 0.05). The clinical pregnancy rate of patients in the letrozole group (53.2 %) was significantly higher than the HRT group (44.4 %, P < 0.05), while no significant difference was observed between the letrozole and natural cycle groups (51.3 %, P > 0.05). Estradiol levels on the day of human chorionic gonadotropin administration in the letrozole group were significantly lower than those in the natural cycle group (280.32 ± 125.39 pg/ml and 351.06 ± 123.03 pg/ml, respectively; P < 0.05). The live birth rate of patients in letrozole group (44.6 %) was significantly higher than the HRT group (32.5 %, P < 0.05), while abortion rate (12.0 %) was significantly lower than the HRT group (21.0 %, P < 0.05). There were no significant differences in number of mature follicles, endometrial thickness, duration of follicle growth between the letrozole and the natural cycle groups, and there were no significant differences in twin birth rate and ectopic pregnancy rate among the three groups (all P values >0.05).

Conclusions

Ovulation induction with letrozole during endometrial preparation for FET has a higher rate of pregnancy success and a lower abortion rate than HRT. Letrozole treatment exhibits clinical progression and outcomes similar to those patients undergoing a natural cycle or normal ovulation cycle. Therefore, letrozole treatment may be an effective option in endometrial preparation for FET in patients with ovulation disorders or irregular menstruation.     

Endometrial thickness influences neonatal birth weight in pregnancies with obstetric complications achieved after fresh IVF–ICSI cycles

Purpose

Pregnancy-associated complications, duration of gestation and parity are well-known predictors of neonatal birth weight. Assisted reproductive technology (ART) affects neonatal birth weight as well. Endometrial thickness as measured on the day of HCG triggering may therefore impact on the neonatal birth weight.

Methods

The data of 764 singleton deliveries achieved after fresh transfer between November 1997 and 2014 were collected retrospectively with the intention to analyze the relationship of maternal and neonatal characteristics with endometrial thickness and the possible predictive value of endometrial thickness on neonatal birth weight.

Results

Higher maternal age (p < 0.001), diminished ovarian reserve (p < 0.001), endometriosis (p = 0.008) and hypogonadotropic hypogonadism (p < 0.001) predicted thin endometrium. Neonatal birth weight (p = 0.004), longer duration of pregnancy (p = 0.008), parity (p = 0.026) and higher maternal BMI (p = 0.003) were correlated significantly with the degree of endometrial proliferation. Endometrial thickness strongly predicted neonatal birth weight (p = 0.004). After adjusting regression analysis for maternal age and BMI, parity, neonatal gender and pregnancy duration, endometrial thickness remained predictive for neonatal birth weight in pregnancies with obstetric complications (p = 0.017). In uneventful pregnancies duration and parity are determinants of neonatal birth weight.

Conclusions

Our findings suggest that endometrial thickness is an additional ART-related factor influencing neonatal birth weight. This finding should be confirmed in large cohort studies.

     

Effect of embryo transfer difficulty on live birth rates studied in vitrified–warmed euploid blastocyst transfers

Research questionThe aim of this study was to investigate the relationship between the different manoeuvres employed or degrees of difficulty during embryo transfer and live birth rate (LBR) in frozen euploid (blastocyst) embryo transfer (FEET).DesignA retrospective, observational study of women undergoing FEET was performed. If the catheter encountered any resistance in its passage through the cervix, a stepwise approach was used. Easy embryo transfer was defined as a direct embryo transfer or use of the outer sheath of the catheter. Difficult embryo transfer was defined as when the process required the use of a Wallace Malleable Stylet (Smiths Medical International Ltd., UK) without or with additional instrumentation such as a tenaculum or uterine sound.ResultsThe analysis involved 370 FEET. LBR was significantly lower in difficult FEET procedures compared with easy ones (54.5% versus 40.5%, P = 0.026) but significance was lost after adjustment for confounders. Use of the outer sheath use did not affect LBR. Although LBR was significantly lower when the stylet, without or with a tenaculum, was required (odds ratio [OR] 0.56, 95% confidence interval [CI] 0.34–0.93; P < 0.05), no statistically significant reduction was observed after adjustment for confounders (OR 0.81, 95% CI 0.45–1.47).ConclusionsNo significant reduction in LBR was observed after adjustment for confounders between difficult and easy FEET, or when use of stylet without or with a tenaculum was required for embryo transfer. The lack of significance may be due to factors such as the sample size or the use of array comparative genomic hybridization analysis. Further studies are needed to confirm these results.     

Characterization of early pregnancy placental progesterone production by use of dydrogesterone in programmed frozen–thawed embryo transfer cycles

Research questionWhen and how does the gradual transition of the endocrine control of early pregnancy from the corpus luteum to the placenta, termed luteoplacental shift, take place?DesignProspective analysis of serum progesterone levels in pregnancies (n = 88) resulting from programmed frozen–thawed embryo transfer cycles in which ovulation was suppressed and no corpus luteum was present. Dydrogesterone, which does not cross-react with progesterone in immunoassay or spectrometric assay, was used for luteal phase and early pregnancy support. Progesterone, oestradiol and hCG were measured at regular intervals from before pregnancy achievement until +65 to 71 days after embryo transfer by Roche Elecsys electrochemiluminescence immunoassay (Elecsys ECLIA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS).ResultsSerum progesterone remained at baseline levels on first blood analysis +9 to 15 days after embryo transfer and increased only marginally independently from the type of pregnancy up to +16 to 22 days after embryo transfer. From +23 to 29 days after embryo transfer, progesterone increased non-linearly above 1.0 ng/ml and increased further throughout the first trimester with elevated levels in multiples. Oestradiol levels increased in parallel with progesterone; hCG plateaued around +37 to 43 days. Progesterone levels were significant predictors for pregnancy viability from +23 to 29 days after embryo transfer onwards with best accuracy +37 to 43 days after embryo transfer (receiver operator characteristic analysis area under the curve 0.98; 95% CI 0.94 to 1; P = 0.0009).ConclusionsThe onset of substantial progesterone production is the 7th gestational week. Progesterone increase is non-linear, depends on chorionicity and zygosity, and may have predictive potential on the outcome of pregnancies originating from frozen embryo transfer cycles.     

The artificial cycle method improves the pregnancy outcome in frozen–thawed embryo transfer: a retrospective cohort study

Abstract

The aim of this retrospective cohort study was to investigate which preparation method is optimal for frozen–thawed embryo transfer (FET) treatment. Analyses were performed on 3160 FET cycles, including 654 cycles with a natural cycle (NC) protocol and 2506 cycles with an artificial cycle (AC) protocol. The primary outcome measures were the clinical pregnancy rate (CPR) and the live birth rate (LBR) per transfer. The Student’s t-test, chi-square test and multiple logistic regression were used for statistical analysis. The CPR per transfer was 49.4% in the NC group and 58.6% in the AC group (OR?=?1.270, 95% CI: 1.037–1.554). The LBR per transfer was 42.2% and 50.8% in the NC and AC groups, respectively (OR?=?1.269, 95% CI: 1.037–1.552). Dividing the patients according to the type of transferred embryos, the CPR (67.3% versus 57.0%, p?<?0.01) and LBR (58.8% versus 49.7%, p?<?0.01) were higher after the AC protocol than after NC protocol in patients with blastocyst transfer. The NC and AC protocols yielded comparable CPR and LBR in the patients with cleavage embryo transfer. Our data indicate better pregnancy outcomes after the AC protocol than after the NC protocol. The AC protocol should be recommended in patients who were counseled before receiving FET treatment. Further studies are needed to confirm this finding.     

Vaginal progesterone supplementation has no effect on ongoing pregnancy rate in hCG-induced natural frozen–thawed embryo transfer cycles

Objective

The purpose of this study is to assess the effect of luteal phase supplementation (LPS) on pregnancy rates in human chorionic gonadotropin (hCG)-induced natural frozen–thawed (FET) cycles.

Study design

All performed hCG-induced natural FET cycles from January 2006 until August 2007 were retrospectively identified. The study group consisted of 452 cycles: 243 supplemented with progesterone administration (600 mg natural micronized progesterone in three separate doses) and 209 without progesterone. Analysis was limited to cycles where embryos were cryopreserved on day 3. Final oocyte maturation was achieved by hCG when endometrial thickness of ≥7 mm and a follicle of 17 mm were present on ultrasound.

Results

No statistically significant differences were observed in ongoing pregnancy rate between the two groups (22% versus 21%, p = 0.8; difference +1%; 95% confidence interval (CI): −6.5 to +8.7). The non-significant effect of the presence or not of luteal support on pregnancy rate was confirmed by logistic regression (odds ratio (OR): 0.9, 95% CI: 0.54–1.47, P = 0.64). A previous pregnancy following fresh embryo transfer (OR: 6.04, 95% CI: 3.63–10.02, P = 0.001) and increased endometrial thickness (OR: 1.25, 95% CI: 1.11–1.41, P = 0.001) significantly affected the achievement of ongoing pregnancy, whereas the association between embryo score and achievement of pregnancy was marginally significant (OR:0.28, 95% CI: 0.08–0.97, P = 0.05).

Conclusion

There is no convincing evidence to support the use of LPS in hCG-induced natural FET cycles, since there is no luteal phase defect. Further prospective randomized studies are necessary to confirm these findings.     

Progesterone for preparation of the endometrium for frozen–thawed blastocyst transfer in vitro fertilization cycles: a prospective study on patients’ opinions on a new subcutaneous formulation

We aimed to evaluate patients’ perspectives on a progesterone subcutaneous formulation for endometrial preparation for frozen-thawed blastocyst transfer. In this prospective study, women with at least one experience with vaginal progesterone, undergone endometrial preparation with oral estradiol valerate and daily subcutaneous progesterone administered from the fifth day before the transfer until the day of the beta-hCG test. Patients completed three questionnaires, at enrollment (Q1), for gathering information on the experience with vaginal treatment and expectations about the subcutaneous route and then at the time of the transfer (Q2) and eight days later (Q3). Main outcome measures were patients' opinions on comfort, ease of use, convenience, overall satisfaction, level of anxiety and pain associated with the administration of subcutaneous progesterone in comparison with their previous experience. Sixty-nine women completed the questionnaires. All vaginal versus subcutaneous comparisons were significantly in favor of the subcutaneous route. When comparing patients’ expectations at Q1 with patients’ opinions at Q2 and Q3, all evaluations, except for one, demonstrated that the patient’s positive expectation was confirmed after 5 and 13 days of treatment. In conclusion, in women with previous experience with vaginal progesterone, the subcutaneous route was associated with significantly increased acceptance.     

IGF2/ApaI polymorphism associated with birth weight in children of the region of Petrolina–PE,Brazil

Insulin-like growth factor 2 (IGF2) gene has an important role in fetal growth. It was investigated association of the IGF2/ApaI polymorphism with low birth weight and normal birth weight (as control) in children attended in Hospital Dom Malan Petrolina, PE-Brazil. The genotype frequencies did not differ statistically between low birth weight (AA = 16.22%, AG = 43.24%, GG = 40.54%) and control (AA = 20% AG = 35%, GG= 45% groups) and the allele frequencies were not significantly different (p > 0.05).The observed genotype frequencies in both groups did not deviate significantly from Hardy–Weinberg equilibrium. Then, no significant correlation was found for this polymorphism in the population studied.     

Is a change in the vaginal flora associated with an increased risk of preterm birth?

OBJECTIVE: The purpose of this study was to determine if a change in the vaginal flora was associated with an increased risk of preterm birth, and to determine if metronidazole therapy before 32 weeks increased the risk of preterm birth. STUDY DESIGN: We compared cultures taken at 23 to 26 weeks of gestation with cultures taken at delivery from women enrolled in the Vaginal Infections and Preterm Birth study to analyze the association of changes in the vaginal flora with preterm birth. RESULTS: Metronidazole therapy before 32 weeks was associated with an increased risk of preterm birth (OR 1.5, 95%CI 1.05-2.1) in an unadjusted model. A change to heavy growth of Escherichia coli or Klebsiella pneumoniae at delivery was found to be associated with preterm birth (OR 2.4, 95%CI 1.6-3.8). After controlling for race, parity, prepregnancy weight <100 pounds, smoking or drinking during pregnancy, Trichomonas vaginalis, bacterial vaginosis, chlamydia, mycoplasmas, group B streptococcus, metronidazole therapy before 32 weeks, vaginal pH >5.0, and an increase in E coli or K pneumoniae , only prepregnancy weight <100 pounds (adjusted odds ratio [AOR] 2.07, 95%CI 1.01-4.21) and increased E coli or K pneumoniae in the vagina at delivery (AOR 2.99, 95%CI 1.37-6.53) were found to be significantly associated with preterm birth. CONCLUSION: An increase in E coli or K pneumoniae in the vagina is an independent risk factor for preterm birth. Changes in the vaginal flora may explain the increased risk of preterm birth seen with vaginal clindamycin or oral metronidazole therapy.     

Extended uterine receptivity for blastocyst implantation and full-term fetal development in mice with vitrified–warmed ovarian tissue autotransplantation

Purpose

Our previous study demonstrated that vitrified?Cwarmed ovarian tissue autotransplantation (VOAT) into estrus cycle-ceased ovariectomized mice restored fertility to achieve full-term fetal development for transferred embryos, while less steroidogenesis in the corpus luteum was observed in VOAT mice. It has been reported that the window of uterine receptivity for blastocyst implantation is extended at lower estrogen levels. Therefore, we hypothesized that duration of the window in VOAT mice could be extended.

Methods

Blastocysts were transferred into VOAT mice on day 5 of pseudopregnancy. Immunohistochemical analysis was performed to examine the potential in VOAT ovarian tissues.

Results

The rate of live birth pups from embryos transferred on day 5 of pseudopregnant VOAT mice was not different from that of embryos transferred on day 4 of pseudopregnancy in VOAT mice, while embryo transfer on day 5 into intact mice showed no pregnancy. Immunohistochemical analysis of the corpus luteum of day 8 pseudopregnant VOAT mice with uteri having decidualization induced on day 5 showed less steroidogenesis and blood vessel formation as compared to intact mice.

Conclusions

Uterine receptivity was extended in VOAT mice. Less steroidogenesis and blood vessel formation in the transferred ovarian tissues may be associated with the extended uterine receptivity.     

Two consecutive successful live birth in woman with 17α hydroxylase deficiency by frozen–thaw embryo transfer under hormone replacement endometrium preparation

17α-Hydroxylase deficiency is rare autosomal recessive disorder that manifested by hypertension, hypokalemia, delayed sexual development, primary amenorrhea and infertility. The information regarding infertility care and conception in women with this disorder are extremely limited. We report a 24-year-old Japanese woman with primary amenorrhea who was diagnosed as partial 17α-hydroxylase deficiency caused by homozygous 3?bp deletion in exon 1 of 17α-hydroxylase gene. In vitro fertilization with controlled ovarian stimulation was carried out and all viable embryo were frozen. During ovarian stimulation, serum progesterone levels were markedly elevated, and endometrial growth was impaired. Utilizing frozen-thaw embryo transfer under hormonal replacement (glucocorticoid, estradiol and progesterone), she had successfully given two consecutive live birth. Women with 17α-hydroxylase deficiency with residual ovarian reserve can afford reproductive success by appropriate diagnosis and treatment by assisted reproductive technology.