Is there a relationship between morphokinetic parameters and neonatal sex in fresh embryo transfers?

To investigate whether morphokinetic parameters differ between male and female embryos in IVF embryos resulting in live births, a retrospective cohort study was undertaken. Files of all live births resulting from a single embryo transfer (SET) cultured in time-lapse incubators between 2013 and 2019 in two tertiary care centres were reviewed. The study group consisted of 187 SETs resulted in 187 live births, of which 100 were females (53.5%) and 87 were males (46.5%). Embryo selection for transfer was based on the known implantation data (KID) score provided by the Embryoscope and morphological assessment by experienced embryologists. Neonatal sex was confirmed through live birth documentation. Morphokinetic parameters and day 3 and day 5 KID scores of male and female embryos were compared. Maternal baseline and treatment characteristics were similar between groups. Morphokinetic time-lapse parameters of male and female embryos including: pronuclei fading; cleavage timings (t2-t9); second and third cell cycle durations; synchrony of the second and third cleavages; late morphokinetic parameters and KID scores did not differ between groups. In conclusion, time-lapse morphokinetic parameters and embryo selection methods do not seem to differ between male and female embryos, and their utilization does not bias towards any neonatal sex.

Does 'Dual Trigger' Increase Oocyte Maturation Rate?

The aim of this study was to evaluate the oocyte maturation rate when GnRH-a and hCG (dual trigger) are co-administered, compared to the standard hCG trigger within the same patient. Included in the study were GnRH antagonist ICSI cycles performed in 137 patients who had a standard hCG trigger cycle and a dual trigger cycle between 1/1/2013 and 31/12/2017. The mean patient age (35.9 ± 5.6 and 35.2 ± 5.9; <0.001), FSH dose (4140 ± 2065 and 3585 ± 1858; <0.01), number of retrieved oocytes (10.3 ± 6.2 and 8.9 ± 6.1; 0.011) were higher in the dual trigger group compared to the hCG trigger group, oocyte maturation rate was identical. Maturation rate following dual trigger was significantly higher among 34 patients who had a maturation rate of <70% following hCG triggering and among 16 patients with a maturation rate <50% rate following hCG trigger (54% vs. 74%, p < .001 and 44% vs. 73%, p = .006; respectively). In conclusion, co-administration of GnRH agonist and hCG for final oocyte maturation substantially increased the oocyte maturation rate in patients with low oocyte maturation rate in their hCG triggered cycle, but not in an unselected population of patients.IMPACT STATEMENTWhat is already known on this subject? The co-administration of GnRH agonist and hCG for final oocyte maturation prior to oocyte retrieval may improve IVF outcome in patients with a high proportion of immature oocytes. The few studies on dual trigger in patients with a high proportion of immature oocytes or in normal responders have shown conflicting results.What do the results of this study add? We found that co-administration of GnRH agonist and hCG for final oocyte maturation substantially increased the oocyte maturation rate in patients with low oocyte maturation rate in their hCG triggered cycle, but not in an unselected population of patients.What are the implications of these findings for clinical practice and/or further research? The results of this study implicate that in selected population with low oocyte maturation rate, there is an advantage in using dual trigger. However, larger prospective trials are warranted to better assess oocyte response in dual trigger.

Morphokinetic characteristics of embryos derived from in-vitro-matured oocytes and their in-vivo-matured siblings after ovarian stimulation.

RESEARCH QUESTION: Does delayed maturation of aspirated metaphase I (MI) oocytes, completed in vitro, adversely affect early embryo development? DESIGN: Time-lapse microscopy was used to compare morphokinetic variables between embryos derived from oocytes with delayed maturation after ovarian stimulation and from in-vivo-matured metaphase II (MII) sibling oocytes from the same IVF and intracytoplasmic sperm injection cycle. RESULTS: A total of 1545 injected oocytes in 169 cycles from 149 patients were included. The in-vitro-matured oocytes had lower normal fertilization rates than the MII aspirated oocytes (50.2% versus 68.0%; P < 0.001). Early key developmental events were significantly delayed in the normally fertilized in-vitro-matured compared with in-vivo-matured oocytes (polar body extrusion: 5.4 ± 3 versus 3.9 ± 1.8 h; P < 0.001; pronuclear fading: 27.2 ± 4.7 versus 25.1 ± 4.2 h; P < 0.001, respectively) and synchrony of the second cell cycle was adversely affected. The proportions of embryos with optimal second cell cycle length and second cell cycle were similar but with fewer top-quality embryos, based on an algorithm, for the delayed in-vitro-matured oocytes compared with their in-vivo-matured sibling oocytes (14% versus 29.1%; P < 0.001). CONCLUSIONS: Embryos derived from oocytes that failed to mature in-vivo in standard treatment after ovarian stimulation may show a different morphokinetic profile from their sibling oocytes aspirated at the MII stage after completing maturation in-vivo.

[LAPAROSCOPICALLY-ASSISTED ULTRASOUND-GUIDED PERCUTANEOUS TRANSABDOMINAL OOCYTE COLLECTION: FERTILITY PRESERVATION IN A 17-YEARS-OLD GIRL WITH VAGINAL EWING SARCOMA].

INTRODUCTION: Options for preserving fertility in children and adolescents with cancer depend on patient age, the available time frame, and the treatment regimen. Ovarian stimulation with mature oocyte preservation is often the optimal method in post-menarcheal adolescents. We describe a case of a 17-year-old girl with vaginal soft-tissue Ewing sarcoma in whom transvaginal oocyte collection for fertility preservation was ruled out by the large tumor. To overcome the limitations of the transabdominal approach, we applied a novel method of laparoscopically-assisted ultrasound-guided percutaneous transabdominal oocyte collection. In this manner, we were able to both perform oophorectomy and obtain superficial and deep ovarian follicles for cryopreservation.

Aspiration of immature oocytes during cesarean section for fertility preservation.

BACKGROUND: In vitro maturation (IVM) of immature oocytes is an important technology for selected clinical indications. We previously described a pregnant woman with a history of renal transplantation who underwent oocyte aspiration during cesarean section (CS) for fertility preservation and future surrogacy. CASE: A 27-year-old pregnant woman was diagnosed with neck rhabdomyosarcoma at 37 weeks' gestation. CS was performed with direct aspiration of small follicles from one ovary and oophorectomy of the other. Twenty-one identified oocyte-cumulus complexes were cultured, and 12 mature oocytes and 14 ovarian cortex strips were cryopreserved. CONCLUSION: Aspirating competent oocytes during CS may serve as an additional means of fertility preservation in pregnant women. The procedure may also be offered to patients with an IVF pregnancy who are scheduled for elective CS.

Prolonging oocyte in vitro culture and handling time does not compensate for a shorter interval from human chorionic gonadotropin administration to oocyte pickup.

OBJECTIVE: To assess the impact of oocyte aspiration, denudation, and sperm injection timing in relation to oocyte hCG exposure time on intracytoplasmic sperm injection (ICSI) outcome. DESIGN: Cohort study. SETTING: Tertiary medical center. PATIENT(S): A total of 614 consecutive ICSI cycles were performed in 421 patients aged <38 years with at least three aspirated oocytes and no more than three previous treatments. INTERVENTION(S): Gonadotropin-releasing hormone agonist or GnRH antagonist suppression; oocyte pickup (OPU)-hCG interval more/less than 36 hours; OPU-denudation interval more/less than 2 hours; denudation-ICSI interval more/less than 1 hour. MAIN OUTCOME MEASURE(S): Fertilization, embryo transfer, and pregnancy rates. RESULT(S): Late OPU was associated with more available embryos than early OPU and significantly higher rates of fertilization (66.0% ± 22.8% vs. 61.8% ± 24.3%), ET (99.5% vs. 96.2%), and pregnancy (47.2% vs. 35.4%). This advantage was more pronounced in GnRH agonist cycles. The length of incubation before or after denudation had no effect, regardless of OPU timing. On logistic stepwise regression, OPU timing was the only significant independent predictor of pregnancy (odds ratio 1.6, 95% confidence interval 1.17-2.29). CONCLUSION(S): The timing of OPU has a predominant effect on ICSI success, especially in GnRH agonist cycles. Delaying oocyte denudation or sperm injection does not compensate for insufficient postpriming exposure to the follicular environment.

Progesterone-to-follicle index is better correlated with in vitro fertilization cycle outcome than blood progesterone level.

OBJECTIVE: To investigate the impact of late follicular phase progesterone (P) elevation in relation to ovarian response on cycle outcome. DESIGN: Cohort study. The progesterone-to-follicle index (PFI) was calculated by dividing the blood P by the number of follicles ≥14 mm. The clinical pregnancy rate was calculated against the range of PFI values and blood P levels. SETTING: In vitro fertilization unit. PATIENT(S): A heterogenous population undergoing IVF with pituitary suppression and gonadotropin stimulation resulting in 3-15 follicles ≥14 mm and blood P≤10 nmol/L on hCG day and resulting in fresh embryo transfer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Association of blood P and PFI with clinical pregnancy rate. RESULT(S): Data were retrieved for 8,649 IVF cycles in normal responders. The (reverse) odd ratios for pregnancy were 1.112 (95% confidence interval [CI], 1.077-1.165) for blood P and 4.104 (95% CI, 3.188-5.284) for the PFI. Elevated P levels were associated with a lower pregnancy rate only when they reached the >93rd percentile. The PFI was inversely and linearly related to the pregnancy rate for the whole range of values. CONCLUSION(S): A late increase in P level is detrimental if it is a consequence of increased P production per follicle (high PFI) but not if it is a consequence of additional follicular recruitment. The PFI enables clinicians to differentiate these conditions.

Pregnancy outcome after ICSI with thawed testicular sperm from men with non-obstructive azoospermia compared to ICSI with ejaculated sperm from men with severe oligoasthenoteratozoospermia and IVF with normal ejaculated sperm.

The aim of the study was to evaluate the clinical pregnancy outcomes, fetal complications and malformation rate of intracytoplasmic injection of thawed cryopreseverd sperm extracted by testicular aspiration from men with non-obstructive azoospermia (NOA) compared with intracytoplasmic injection of fresh ejaculated sperm from men with severe oligoteratoasthenozoospermia (OTA) and standard in vitro fertilization using ejaculated sperm from normospermic men. The mean oocyte fertilization rate was significantly lowest for ICSI with testicular aspirated sperm (NOA group). However, there was no significant difference among the three groups in pregnancy outcomes, namely rates of spontaneous abortion, biochemical pregnancy, extrauterine pregnancy, singleton multifetal pregnancy, preterm delivery before 36 weeks' gestation, maternal complications, transfer to the neonatal intensive care unit, intrauterine growth restriction or fetal malformations. These results suggest that despite some earlier findings that intracytoplasmic injection of aspirated sperm from men with NOA is associated with lower fertilization rates and embryo quality, the pregnancy and immediate neonatal outcomes may be unaffected.

Growth differentiating factor 9 (GDF9) and bone morphogenetic protein 15 both activate development of human primordial follicles in vitro, with seemingly more beneficial effects of GDF9.

CONTEXT: The signals initiating growth of primordial follicles are unknown. Bone morphogenetic protein 15 (BMP15) and growth differentiating factor 9 (GDF9) are promising candidates. OBJECTIVE: The objective of the study was to evaluate for the first time the effects of human recombinant BMP15 and human recombinant GDF9 on the in vitro development of human primordial follicles. DESIGN AND SETTING: This was a controlled culture study performed in a major tertiary university-affiliated medical center. MATERIALS: Materials included ovarian tissue from 17 girls/women and three aborted human fetuses. INTERVENTION: There were no interventions. MAIN OUTCOME MEASURE: Histological and immunohistochemical (proliferating cell nuclear antigen, BMP15, and GDF9) studies and an endocrine assay of 17ß-estradiol were conducted. RESULTS: In the samples from girls/women, the number of developing follicles was greater with GDF9 or BMP15 alone than with no BMP15 or GDF9. Higher 17ß-estradiol secretion was noted after treatment with GDF9 than with BMP15 or with GDF9+anti-GDF9. The number of atretic follicles was greater with BMP15 than with GDF9. Proliferating cell nuclear antigen expression was greater with the higher dose of both growth factors than the lower dose. Expression of BMP15 and GDF9 was identified in samples cultured without BMP15 or GDF9. Results for the fetal follicles yielded no distinguishable pattern. CONCLUSIONS: Although both BMP15 and GDF9 promoted activation of human primordial follicles from girls/women (but not human fetuses) in a dose-dependent manner, GDF9 seems more beneficial.

Neurotrophin 4 enhances in vitro follicular assembly in human fetal ovaries.

OBJECTIVE: To investigate the in vitro effect of neurotrophin 4 (NT-4) on follicular assembly in human fetal ovaries. DESIGN: Human ovarian tissue from fetuses at 19-20 gestational weeks was placed in organ culture for two weeks with NT-4. Control groups were cultured with a neutralizing antibody against NT-4. SETTING: Infertility unit at an university-affiliated tertiary medical center. PATIENT(S): Four patients who underwent pregnancy terminations at 19-20 gestational weeks. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Histologic findings of follicular assembly. RESULT(S): Follicular assembly was significantly increased in the specimens cultured with NT-4 than in the uncultured specimens, the samples cultured without NT-4, and samples cultured with the neutralizing antibody. In the second week of culture, additional follicular assembly was promoted in the presence of 100 ng/mL NT-4 but not with 10 ng/mL NT-4. CONCLUSION(S): This is the first report showing that NT-4 seems to promote human follicular assembly in fetal ovaries, probably in a dose-dependent manner. Follicular assembly is regulated by multiple signals, and additional studies on the effects of other growth factors in combination with NT-4 are warranted.

WITHDRAWN: Bone Morphogenetic Protein 15 (BMP15)/Growth Differentiating Factor 9 Heterodimer Is More Potent than BMP15 Alone in in Vitro Activation of Human Primordial Follicles.

This manuscript was withdrawn at the request of the authors.

Glial cell line-derived neurotrophic factor (GDNF) and its receptors in human ovaries from fetuses, girls, and women.

OBJECTIVE: To investigate the expression of glial cell line-derived neurotrophic factor (GDNF) and its receptors, GDNF alpha 1 (GFR-alpha1) and tyrosine kinase receptor for rearranged during transfection (RET), in human ovaries at the protein and mRNA levels. DESIGN: Samples were prepared for immunohistochemical staining for GDNF, GFR-alpha1, and RET and in situ hybridization for the mRNA of GFR-alpha1. The mRNA expression of two GDNF isoforms and two RET isoforms was investigated by reverse transcription polymerase chain reaction. SETTING: Infertility unit at a university-affiliated tertiary medical center. PATIENT(S): Fifteen patients who underwent pregnancy terminations at 21-35 gestational weeks and 28 girls/women aged 5-39 years who underwent ovarian laparoscopies. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Laboratory analysis of human ovarian specimens. RESULT(S): The GDNF protein was detected in oocytes of all samples. Granulosa cells (GCs) stained for GDNF in a portion of fetal samples and in all samples from girls/women. The proteins for GFR-alpha1 and RET were detected in both oocytes and GCs with weak staining for the long RET isoform. GFR-alpha1 mRNA transcripts were detected in oocytes and GCs from all samples. The mRNA transcripts for the two GDNF isoforms and the two RET isoforms were detected in all fetal and adult specimens. CONCLUSION(S): The presence of the receptors of GDNF in the GCs of human primordial follicles suggests that GDNF may be involved in the regulation of primordial follicular activation.

Vascular endothelial growth factor A and its two receptors in human preantral follicles from fetuses, girls, and women.

OBJECTIVE: To investigate the expression of vascular endothelial growth factor A (VEGF-A) and that its two receptors (VEGFR1, VEGFR2) in human preantral follicles. DESIGN: Immunohistochemical, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) study of the expression of the VEGF-A system in human ovaries. SETTING: Major tertiary-care academic center. PATIENT(S): Twenty-two patients who underwent pregnancy terminations at 21-35 gestational weeks and 29 girls/women aged 5-39 years who underwent ovarian laparoscopies. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Laboratory analysis of human ovarian specimens. RESULT(S): Immunhistochemistry and in situ hybridization revealed the expression of the proteins and mRNA transcripts for VEGFR1 and VEGFR2 in oocytes, granulosa cells, and stroma cells from fetuses and girls/women. The protein for VEGF-A was detected immunohistochemically in oocytes, granulosa cells, and stroma cells from fetuses and girls. VEGF-A and VEGFR1 proteins were expressed more strongly than VEGFR2. VEGF-A(121), VEGF-A(165), and VEGF-A(189) isoforms were identified by RT-PCR in the ovarian samples from fetuses and women. CONCLUSION(S): The presence of the VEGF-A receptors, particularly in the granulosa cells, suggests that VEGF-A might be involved in proliferation initiation of primordial follicles or play an as yet unknown role in human preantral follicles.

Effects of basic fibroblast growth factor on in vitro development of human ovarian primordial follicles.

OBJECTIVE: To evaluate whether basic fibroblast growth factor (FGF) benefits the in vitro development of human primordial follicles. DESIGN: Human ovarian tissue was placed in organ culture for 4 weeks with basic FGF and either fetal calf serum or a serum-free combination. Control groups were cultured with a neutralizing antibody against basic FGF. SETTING: Major tertiary care and referral academic centers. PATIENT(S): Fourteen women/girls undergoing various gynecological operations and two fetuses from women undergoing pregnancy terminations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicular counts, immunohistochemistry for proliferating cell nuclear antigen and bromodeoxyuridine incorporation and measurement of 17beta E(2) production. RESULT(S): Only in the serum-free culture system was the number of developing follicles in samples cultured with basic FGF significantly higher than in uncultured specimens. The E(2) production increased significantly in the second week, and there was a significant reduction in E(2) secretion with the addition of the neutralizing antibody against basic FGF. The percentage of granulosa cells (GCs) that stained for proliferating cell nuclear antigen or bromodeoxyuridine was significantly higher in developing follicles than in primordial follicles, regardless of treatment. CONCLUSION(S): Basic FGF apparently plays a role in the E(2) production of early follicles. High doses of basic FGF enhanced follicular development in serum-free media.

Transplantation of frozen-thawed late-gestational-age human fetal ovaries into immunodeficient mice.

OBJECTIVE: To compare the development of human fetal follicles from late-gestational-age fetuses frozen-thawed gradually and slowly with dimethylsulfoxide (DMSO) or 1,2 propanediol (PROH) and sucrose after renal grafting into follicle stimulating hormone-treated immunodeficient mice. DESIGN: Controlled histologic study of grafted human fetal ovaries. SETTING: Major tertiary care academic center. PATIENT(S): Eleven women undergoing pregnancy termination at 22 to 33 gestational weeks. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Microscopic morphometric analysis and immunohistochemistry for proliferating cell nuclear antigen (PCNA). RESULT(S): Only follicles from samples frozen-thawed with PROH developed to secondary and antral stages 4 to 6 months after grafting, with PCNA expression in their granulosa cells. However, the number of surviving/developing follicles per section was very low (4-25 per graft), compared with 71 to 406 follicles in pretransplantation samples. Graft recovery was very high, with similar rates for transplants frozen-thawed with PROH and DMSO. Normal ovarian structure after grafting was identified only in the PROH frozen-thawed grafts. In deteriorated grafts, frozen-thawed with either DMSO or PROH, net-like hollows replaced follicles, whereas tubule-like structures were only identified in DMSO frozen-thawed grafts. CONCLUSION(S): This is the first report of the development of late-pregnancy-stage human fetal follicles in immunodeficient mice. PROH freezing-thawing supported development and survival better than DMSO. However, the low follicular survival points to the urgent need for efficient methods to enhance vascularization rate and prevent ischemia.

Growth hormone and its receptor in human ovaries from fetuses and adults.

OBJECTIVE: To investigate the presence of growth hormone (GH) and its receptor (GH-R) in early developing follicles. DESIGN: Immunocytochemical and in situ hybridization study. SETTING: Major tertiary care and referral academic centers. PATIENT(S): Ten ovarian samples from adults/girls aged 6-38 years and from 10 fetuses of women undergoing second and third trimester pregnancy terminations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Immunocytochemistry and in situ hybridization on paraffin sections of human ovaries from fetuses and women/girls. RESULT(S): The proteins and the mRNA transcripts for GH and GH-R were detected in oocytes, granulosa (GC), and stroma cells from both sources (fetuses and women/girls), with low staining intensity only in a portion of the fetal GC. CONCLUSION(S): This is the first report of the expression of GH-R in human ovaries from fetuses as well as women/girls and of GH in human fetal ovaries. Be that as it may, further experiments should be conducted to elucidate if indeed GH is involved in the initiation of human primordial follicular growth.