Impact of multinucleated blastomeres on embryo developmental competence, morphokinetics, and aneuploidy.

OBJECTIVE: To study the effect of human embryo multinucleation on the rate of aneuploidy, in vitro developmental morphokinetics, and pregnancy outcome. DESIGN: Retrospective study. SETTING: University-affiliated fertility center. PATIENT(S): A total of 296 patients undergoing IVF cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Rate of multinucleation at the 2- and 4-cell stage, time-lapse morphokinetic parameters from zygote to blastocyst stage, ploidy of embryos analyzed by means of trophectoderm biopsy and array comparative genomic hybridization (PGS), and pregnancy outcome. RESULT(S): A total of 1,055 out of 2,441 (43.2%) embryos evaluated with the use of the Embryoscope time-lapse system showed blastomere multinucleation at the 2-cell stage (MN2). The frequency of this abnormality was substantially reduced in 4-cell-embryos (15.0%). Among all clinical factors analyzed, only maternal age had a positive correlation with multinucleation rate. The timing of cleavage divisions from the pronuclear fading to 5-cell embryo was significantly longer (1.0-2.5 h) in MN2 embryos than in non-MN2 control samples. Of the total embryos tested with the use of PGS (n = 607), the rates of multinucleation were similar in euploid versus aneuploid blastocysts (40.8% and 46.7%, respectively). All 24 chromosomes contributed to aneuploidy of MN2 embryos. There were 61 transfers of MN2 embryos that resulted in 45.9% clinical pregnancies and a 31.6% implantation rate. CONCLUSION(S): The frequency of multinucleation is high in human embryos cultured in vitro and equally affects euploid and aneuploid human embryos. It appears that most MN embryos have the capacity for self-correction during early cleavage divisions and can develop into euploid blastocysts resulting in healthy babies.

Assisted reproduction involving gestational surrogacy: an analysis of the medical, psychosocial and legal issues: experience from a large surrogacy program.

STUDY QUESTION: What are the medical, psychosocial and legal aspects of gestational surrogacy (GS), including pregnancy outcomes and complications, in a large series? SUMMARY ANSWER: Meticulous multidisciplinary teamwork, involving medical, legal and psychosocial input for both the intended parent(s) (IP) and the gestational carrier (GC), is critical to achieve a successful GS program. WHAT IS KNOWN ALREADY: Small case series have described pregnancy rates of 17-50% for GS. There are no large case series and the medical, legal and psychological aspects of GS have not been addressed in most of these studies. To our knowledge, this is the largest reported GS case series. STUDY DESIGN, SIZE AND DURATION: A retrospective cohort study was performed. Data were collected from 333 consecutive GC cycles between 1998 and 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: There were 178 pregnancies achieved out of 333 stimulation cycles, including fresh and frozen transfers. The indications for a GC were divided into two groups. Those who have 'failed to carry', included women with recurrent implantation failure (RIF), recurrent pregnancy loss (RPL) and previous poor pregnancy outcome (n = 96; 132 cycles, pregnancy rate 50.0%). The second group consisted of those who 'cannot carry' including those with severe Asherman's syndrome, uterine malformations/uterine agenesis and maternal medical diseases (n = 108, 139 cycles, pregnancy rate 54.0%). A third group, of same-sex male couples and single men, were analyzed separately (n = 52, 62 cycles, pregnancy rate 59.7%). In 49.2% of cycles, autologous oocytes were used and 50.8% of cycles involved donor oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: The 'failed to carry' group consisted of 96 patients who underwent 132 cycles at a mean age of 40.3 years. There were 66 pregnancies (50.0%) with 17 miscarriages (25.8%) and 46 confirmed births (34.8%). The 'cannot carry pregnancy' group consisted of 108 patients who underwent 139 cycles at a mean age of 35.9 years. There were 75 pregnancies (54.0%) with 15 miscarriages (20.0%) and 56 confirmed births (40.3%). The pregnancy, miscarriage and live birth rates between the two groups were not significantly different (P = 0.54; 0.43; 0.38, respectively). Of the 178 pregnancies, 142 pregnancies were ongoing (surpassed 20 weeks) or had ended with a live birth and the other 36 pregnancies resulted in miscarriage (25.4%). Maternal (GS) complication rates were low, occurring in only 9.8% of pregnancies. Fetal anomalies occurred in only 1.8% of the babies born. LIMITATIONS, REASONS FOR CAUTION: Although it is a large series, the data are retrospective and conclusions must be drawn accordingly while considering bias, confounding and power. Due to the retrospective nature of this study, follow-up data on 6.3% of birth outcomes were incomplete. In addition, long-term follow-up data on GCs and IPs were not available to us at the time of publication. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the largest GS series published. We have included many details regarding not only the medical protocol but also the counseling and legal considerations, which are an inseparable part of the process. Data from this study can be included in discussions with future intended parents and gestational carriers regarding success rates and complications of GS.

Comparison of IVF Outcomes between Minimal Stimulation and High-Dose Stimulation for Patients with Poor Ovarian Reserve.

We examined whether treatment with minimum-dose stimulation (MS) protocol enhances clinical pregnancy rates compared to high-dose stimulation (HS) protocol. A retrospective cohort study was performed comparing IVF and pregnancy outcomes between MS and HS gonadotropin-antagonist protocol for patients with poor ovarian reserve (POR). Inclusion criteria included patients with an anti-Müllerian hormone (AMH) ≤8 pmol/L and/or antral follicle count (AFC) ≤5 on days 2-3 of the cycle. Patients from 2008 exclusively had a HS protocol treatment, while patients in 2010 had treatment with a MS protocol exclusively. The MS protocol involved letrozole at 2.5 mg over 5 days, starting from day 2, overlapping with gonadotropins, starting from the third day of letrozole at 150 units daily. GnRH antagonist was introduced once one or more follicles reached 14 mm or larger. The HS group received gonadotropins (≥300 IU/day) throughout their antagonist cycle. Clinical pregnancy rate was significantly higher in the MS protocol compared to the HS protocol (P = 0.007). Furthermore, the live birth rate was significantly higher in the MS group compare to the HS group (P = 0.034). In conclusion, the MS IVF protocol is less expensive (lower gonadotropin dosage) and resulted in a higher clinical pregnancy rate and live birth rate than a HS protocol for poor responders.

Is the zona pellucida thickness of human embryos influenced by women's age and hormonal levels?

OBJECTIVE: To evaluate whether zona pellucida thickness (ZPT) of human embryos is correlated with maternal age, patient's hormonal status, embryo quality, and IVF outcomes. DESIGN: Prospective study. SETTING: University-affiliated IVF clinic. PATIENT(S): Couples undergoing IVF-ET cycles. INTERVENTION(S): Zona measurements, clinical data collection. MAIN OUTCOME MEASURE(S): Correlation between the ZPT and maternal age, basal FSH and E(2) levels, stimulation protocols, cause of infertility, embryo quality, and implantation/pregnancy rates. RESULT(S): The measurements of ZPT were collected from 5,184 day 3 human embryos originated from 744 IVF patients. The overall mean ZPT was 16.18 ± 2.00 µm. No significant correlation was observed between the ZPT and the patient's age, E(2) values on the day of hCG administration, basal concentration of serum FSH, stimulation protocol, infertility diagnosis, and implantation/pregnancy rates. The ZPT was strongly influenced only by the embryo quality: Embryos with good morphology exhibited considerably thinner ZP compared with those of less favorable morphology (mean 15.87 ± 2.48 µm vs. 16.36 ± 2.57 µm, respectively). The ZPT had no significant impact on the implantation and pregnancy rates. CONCLUSION(S): The thickness of the human ZP of day 3 embryos is not influenced by women's age and hormonal levels. The strong correlation between ZPT and embryo quality suggests that thickness of ZP depends on inherent embryo properties. The overall ZPT is not a good predictive indicator for IVF clinical outcomes.

Laser zona thinning in women aged

The objective of this prospective randomized double-blind clinical trial was to evaluate whether laser zona pellucida thinning of human embryos improves clinical outcomes in women

Time-dependent capability of human oocytes for activation and pronuclear formation during metaphase II arrest.

BACKGROUND: The purpose of this study was to investigate the fertilization rate and developmental potential of human oocytes in relation to the duration of their metaphase II (MII) arrest stage following the extrusion of the first polar body (1PB). METHODS: Immature metaphase I oocytes (MI; study oocytes, n = 468) that underwent meiotic maturation during brief in vitro culture and their matured in vivo, MII siblings (control oocytes, n = 3293) were subjected to ICSI. Fertilization and early cleavage were evaluated in both study and control groups. RESULTS: The overall fertilization rate was significantly lower in the oocytes matured in vitro than in those matured in vivo (42 versus 77%, P < 0.0001). A significant relationship was observed between oocyte activation potential and the length of MII arrest. The majority of study oocytes injected soon after PB extrusion remained unfertilized (64%; 98/154 oocytes). The proportion of normally activated oocytes that contained two pronuclei and two PBs gradually increased with prolonged time of MII arrest (43 and 61% at 2 and 3-6 h after 1PB extrusion). Significantly more embryos originating from the study than control oocytes were arrested soon after the first two cleavage divisions (39 and 17%; P < 0.0001) and exhibited multinucleated blastomeres (23 and 13%; P < 0.0001), which suggests the existence of chromosomal abnormalities. CONCLUSIONS: Human oocytes progressively develop the ability for full activation and normal development during the MII arrest stage.

Calcium-binding proteins and calcium-release channels in human maturing oocytes, pronuclear zygotes and early preimplantation embryos.

BACKGROUND: The study aim was to investigate the presence and localization of Ca2+-binding proteins and Ca2+-release receptor channels in human maturing oocytes, pronuclear zygotes and preimplantation embryos. METHODS: Immunocytochemical analysis, using specific antibodies against the proteins being studied, followed with confocal laser microscopy, was performed on human oocytes and embryos. RESULTS: Calreticulin and calsequestrin (the two major calcium storage proteins of somatic cells), two types of calcium release receptors, the inositol trisphosphate and ryanodine receptors (InsP(3)R-2, RyRs-1,2,3), and the molecular chaperone, calnexin, were identified in all investigated cell types. Calreticulin was predominant in the cell cortex and in the nuclear envelope, while calsequestrin was distributed throughout the entire cytoplasm. Generally, localization of the InsP(3)R-2 and RyRs was similar to that of calreticulin and calsequestrin respectively. Both types of receptor were enriched in the subplasmalemmal region of meiotic oocytes. In addition, the InsP(3)R was detected in the nuclear structures of oocytes and blastomeres. Calnexin distribution overlapped with that of calreticulin but appeared to be present in distinct subcompartments. CONCLUSIONS: Human oocytes and embryos express the calcium sequestration and release proteins in highly organized and developmentally regulated patterns. Fine-tuning of these proteins may play a crucial role in regulation of Ca2+ transience during oocyte maturation, fertilization and early embryo development.

Morphological and cytogenetic analysis of human giant oocytes and giant embryos.

BACKGROUND: Giant binuclear oocytes occur with considerable frequency in human ovaries, but their ultimate fate remains unknown. We report the morphology, cytogenetics and developmental potential of human giant oocytes from patients undergoing assisted reproductive technologies. METHODS AND RESULTS: A total of 44 giant oocytes was collected from patients aged 22-44 years old, with an overall frequency of 0.3% (44/14 272 oocytes). Giant oocytes were approximately 30% larger in diameter than normal oocytes (mean 200.4 versus 154.7 micro m, P = 0.0001). Two different morphological patterns were observed among giant unfertilized and fertilized oocytes. All unfertilized oocytes appeared to be diploid and contained either one or two metaphase plates (46 or 2 x 23 chromosomes), and one or two polar bodies respectively. Consequently, fertilized giant oocytes exhibited either two or three pronuclei, or two or four polar bodies. Both types of giant zygotes were capable of normal cleavage and development to blastocyst stage. Four giant embryos were analysed by interphase fluorescence in-situ hybridization using probes for chromosomes 9, 22, X and Y, and all appeared chromosomally abnormal with numerical alterations indicative of ploidy change. CONCLUSIONS: Giant oocytes might be a possible source of human digynic triploidy. To avoid undesired miscarriages, giant embryos originated from either two- or three-pronuclear giant zygotes should be excluded from uterine transfers.