Associations between embryo grading and congenital malformations in IVF/ICSI pregnancies.

RESEARCH QUESTION: Does the quality of transferred embryos have an impact on the rate of congenital malformations in IVF/intracytoplasmic sperm injection (ICSI)-conceived babies? DESIGN: Retrospective cohort study involving 6637 pregnancies of ≥20 weeks' gestation from women undergoing embryo transfer with a single Day 5 embryo at a private multisite IVF clinic between 2005 and 2015. Embryos were classified as good quality (n = 5537) or poor quality (n = 1100) based on an internal grading system of morphological parameters; malformation rates were compared. RESULTS: In pregnancies proceeding to delivery (≥20 weeks' gestation), poor quality embryos were associated with increased odds of at least one anomaly (adjusted odds ratio [OR] 1.33, 95% confidence interval [CI] 1.03-1.71), major anomalies (adjusted OR 1.42, 95% CI 1.05-1.91), musculoskeletal anomalies (adjusted OR 2.09, 95% CI 1.35-3.22), particularly talipes (adjusted OR 2.88, 95% CI 1.33-6.25), and the International Classification of Diseases (ICD) classification 'Other congenital malformations' (adjusted OR 2.34, 95% CI 1.13-4.34). Furthermore, for pregnancies ≥9 weeks' gestation, poor embryos had more than double the odds of chromosomal anomalies than good embryos (adjusted OR 2.33, 95% CI 1.30-4.18, P = 0.005). CONCLUSIONS: This is the first study to compare the rates of individual congenital malformations for good and poor quality embryos. It provides insight into potential risks of transferring poor quality embryos. In pregnancies ≥20 weeks' gestation, poor quality Day 5 embryos are associated with major malformations, at least one anomaly, musculoskeletal anomalies, talipes and the ICD classification 'Other congenital malformations'. In pregnancies ≥9 weeks' gestation, poor quality Day 5 embryos are associated with chromosomal anomalies.

Progesterone concentrations and dosage with frozen embryo transfers - What's best?

BACKGROUND: There is a lack of consensus on the optimal dose and form of progesterone supplementation during frozen-thawed embryo transfer with hormone replacement therapy. AIMS: We aim to identify the serum progesterone concentration on day 16 most likely to result in positive pregnancy outcomes. MATERIALS AND METHODS: We undertook a retrospective study of 4582 women who underwent frozen embryo transfer with hormone replacement therapy, or natural frozen embryo transfer, over 14 years at a multi-site private in vitro fertilisation clinic. Embryos were 3-5 days of age at time of transfer. We extracted data on serum progesterone concentrations and outcomes, as well as dose and form of progesterone supplementation, from patient and pharmacy records. RESULTS: Increased live birth rates for frozen embryo transfer with hormone replacement therapy were seen with day 16 serum progesterone concentrations >50 nmol/L (26.4% vs 11.3% for <50 nmol/L; adjusted odds ratio (OR) 3.14 (95% CI 2.21-4.48)). Similarly, a decreased pregnancy loss rate was seen in this group (14.3% vs 32.6% for ≤50 nmol/L; adjusted OR 0.26 (95% CI 0.12-0.58)). There was a positive correlation between live births and the number of progesterone doses per day (r = 0.119, P = 0.026) and day 16 progesterone concentrations (r = 0.128, P = 0.011). CONCLUSION: Improved pregnancy outcomes are seen with day 16 serum progesterone concentrations >50 nmol/L. There is a statistically significant correlation between live births, number of progesterone doses per day and day 16 serum progesterone concentrations in this study.

Human Blastocyst Secreted microRNA Regulate Endometrial Epithelial Cell Adhesion.

Successful embryo implantation requires synchronous development and communication between the blastocyst and the endometrium, however the mechanisms of communication in humans are virtually unknown. Recent studies have revealed that microRNAs (miRs) are present in bodily fluids and secreted by cells in culture. We have identified that human blastocysts differentially secrete miRs in a pattern associated with their implantation outcome. miR-661 was the most highly expressed miR in blastocyst culture media (BCM) from blastocysts that failed to implant (non-implanted) compared to blastocysts that implanted (implanted). Our results indicate a possible role for Argonaute 1 in the transport of miR-661 in non-implanted BCM and taken up by primary human endometrial epithelial cells (HEECs). miR-661 uptake by HEEC reduced trophoblast cell line spheroid attachment to HEEC via PVRL1. Our results suggest that human blastocysts alter the endometrial epithelial adhesion, the initiating event of implantation, via the secretion of miR, abnormalities in which result in implantation failure.

Prevention of Ovarian Hyperstimulation Syndrome: A Review.

The following review aims to examine the available evidence to guide best practice in preventing ovarian hyperstimulation syndrome (OHSS). As it stands, there is no single method to completely prevent OHSS. There seems to be a benefit, however, in categorizing women based on their risk of OHSS and individualizing treatments to curtail their chances of developing the syndrome. At present, both Anti-Müllerian Hormone and the antral follicle count seem to be promising in this regard. Both available and upcoming therapies are also reviewed to give a broad perspective to clinicians with regard to management options. At present, we recommend the use of a "step-up" regimen for ovulation induction, adjunct metformin utilization, utilizing a GnRH agonist as an ovulation trigger, and cabergoline usage. A summary of recommendations is also made available for ease of clinical application. In addition, areas for potential research are also identified where relevant.

Cryopreserved oocytes: update on clinical applications and success rates.

IMPORTANCE: Over the past 3 decades, oocyte cryopreservation procedures have improved rapidly. However, there is limited research reviewing the efficacy of different cooling protocols and inadequate data comparing in vitro fertilization (IVF) outcomes from fresh oocytes with cryopreserved oocytes. OBJECTIVE: The present review was performed to investigate advances in oocyte cryopreservation technologies and identify areas for further research, to determine whether results from IVF using cryopreserved oocytes are comparable to IVF using fresh oocytes, and to identify the patient populations requiring access to oocyte cryopreservation. EVIDENCE ACQUISITION: A literature review was conducted. OVID (MEDLINE) and PubMed databases were queried using phrases such as "oocyte or egg" and "cryopreservation," "vitrification," or "slow cooling or slow freezing." A total of 180 studies were selected for review. RESULTS: Current literature suggests that vitrified oocytes produce superior IVF results to slow-frozen oocytes and may yield comparable outcomes to IVF with fresh oocytes in certain patient populations. Patients at risk of infertility due to disease or age-related decline or oocyte donation programs, couples who fail to produce semen when required for IVF, and patients with legal or ethical reasons against embryo cryopreservation may access cryopreserved oocytes. CONCLUSIONS: We suggest that women who comprise the previously mentioned patient populations should be offered oocyte vitrification technology. Further research is required to confirm IVF success across all patient populations and determine the best cryopreservation protocols. RELEVANCE: This review will be relevant to clinicians interested in fertility treatments using cryopreserved oocytes, fertility preservation, oncology and fertility, and immunology and fertility.

A pilot double-blind randomised placebo-controlled dose-response trial assessing the effects of melatonin on infertility treatment (MIART): study protocol.

INTRODUCTION: High levels of oxidative stress can have considerable impact on the outcomes of in vitro fertilisation (IVF). Recent studies have reported that melatonin, a neurohormone secreted from the pineal gland in response to darkness, has significant antioxidative capabilities which may protect against the oxidative stress of infertility treatment on gametes and embryos. Early studies of oral melatonin (3-4 mg/day) in IVF have suggested favourable outcomes. However, most trials were poorly designed and none have addressed the optimum dose of melatonin. We present a proposal for a pilot double-blind randomised placebo-controlled dose-response trial aimed to determine whether oral melatonin supplementation during ovarian stimulation can improve the outcomes of assisted reproductive technology. METHODS AND ANALYSES: We will recruit 160 infertile women into one of four groups: placebo (n=40); melatonin 2 mg twice per day (n=40); melatonin 4 mg twice per day (n=40) and melatonin 8 mg twice per day (n=40). The primary outcome will be clinical pregnancy rate. Secondary clinical outcomes include oocyte number/quality, embryo number/quality and fertilisation rate. We will also measure serum melatonin and the oxidative stress marker, 8-hydroxy-2'-deoxyguanosine at baseline and after treatment and levels of these in follicular fluid at egg pick-up. We will investigate follicular blood flow with Doppler ultrasound, patient sleepiness scores and pregnancy complications, comparing outcomes between groups. This protocol has been designed in accordance with the SPIRIT 2013 Guidelines. ETHICS AND DISSEMINATION: Ethical approval has been obtained from Monash Health HREC (Ref: 13402B), Monash University HREC (Ref: CF14/523-2014000181) and Monash Surgical Private Hospital HREC (Ref: 14107). Data analysis, interpretation and conclusions will be presented at national and international conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ACTRN12613001317785.

Localisation of the Notch family in the human endometrium of fertile and infertile women.

To investigate the spatial and temporal immunolocalisation and staining intensity of the Notch signalling family in endometrium of fertile and infertile women, endometrial biopsies were collected by curettage from 25 fertile women across the menstrual cycle and 10 infertile women in the mid secretory phase of menstrual cycle. Immunohisotchemistry was completed for NOTCH1, -2, -3, -4, cleaved Notch, DLL1, -3, -4, JAGGED1, -2, HES and NUMB and immunostaining intensity measured in both the endometrial glandular and luminal epithelium. NOTCH1 and the ligands DLL1 and JAGGED1 were key proteins displaying increased staining intensity during the receptive phase of the menstrual cycle and dysregulated in infertile endometrium. Conversely, NUMB a negative regulator of Notch signalling was decreased in the mid secretory phase of the menstrual cycle in fertile women and increased with infertility.

Fresh versus frozen embryo transfer: backing clinical decisions with scientific and clinical evidence.

BACKGROUND: Improvements in vitrification now make frozen embryo transfers (FETs) a viable alternative to fresh embryo transfer, with reports from observational studies and randomized controlled trials suggesting that: (i) the endometrium in stimulated cycles is not optimally prepared for implantation; (ii) pregnancy rates are increased following FET and (iii) perinatal outcomes are less affected after FET. METHODS: This review integrates and discusses the available clinical and scientific evidence supporting embryo transfer in a natural cycle. RESULTS: Laboratory-based studies demonstrate morphological and molecular changes to the endometrium and reduced responsiveness of the endometrium to hCG, resulting from controlled ovarian stimulation. The literature demonstrates reduced endometrial receptivity in controlled ovarian stimulation cycles and supports the clinical observations that FET reduces the risk of ovarian hyperstimulation syndrome and improves outcomes for both the mother and baby. CONCLUSIONS: This review provides the basis for an evidence-based approach towards changes in routine IVF, which may ultimately result in higher delivery rates of healthier term babies.

Fetal-maternal communication: the role of Notch signalling in embryo implantation.

The establishment of a successful pregnancy requires the implantation of a competent blastocyst into a 'receptive' endometrium, facilitating the formation of a functional placenta. Inadequate or inappropriate implantation and placentation is a major reason for infertility and is thought to lead to first-trimester miscarriage, placental insufficiency and other obstetric complications. Blastocyst-endometrial interactions are critical for implantation and placental formation. The Notch signalling family is a receptor-ligand family that regulates cellular processes as diverse as proliferation, apoptosis, differentiation, invasion and adhesion. Notch signalling is achieved via cell-cell interaction; thus, via Notch, cells can have direct effects on the fate of their neighbours. Recently, a number of studies have identified Notch receptors and ligands in the endometrium, blastocyst and placenta. This review collates current knowledge of this large receptor-ligand family and explores the role of Notch signalling during implantation and placentation, drawing on information from both human and animal studies. Overall, the evidence suggests that Notch signalling is a critical component of fetal-maternal communication during implantation and placentation and that abnormal Notch expression is associated with impaired placentation and pre-eclampsia.

Supersensitive fluorescent semen analysis: validation on azoospermic and oligozoospermic samples.

OBJECTIVE: To compare the clinical utility of a supersensitive fluorescent semen analysis (SSSA) procedure published by Cooper et al. (2006) with a conventional World Health Organization (WHO)-based semen analysis technique in males with severe oligozoospermia or azoospermia who are undergoing fertility assessment. DESIGN: Prospective single-center study. SETTING: IVF clinic. PATIENT(S): Patients attending an infertility clinic for semen analysis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Presence of spermatozoa in the ejaculate. RESULT(S): Semen samples from 100 men were analyzed using conventional WHO 4th Edition semen analysis and determined to be either severely oligozoospermic or azoospermic (reported lower limit of detection of 0.1 million sperm/mL). An aliquot of the same unprocessed sample was also analyzed using the SSSA protocol (reported lower limit of detection of approximately 8000 sperm/mL). The SSSA method confirmed the results of conventional semen analysis in 77% of cases. In 22% of cases, sperm were identified only using SSSA. Overall, SSSA was capable of identifying the presence of sperm in significantly more samples than conventional semen analysis. CONCLUSION(S): The reliable differentiation of extreme oligospermia from azoospermia has profound implications in fertility management. This paper provides the first data comparing sperm detection rates using SSSA or conventional WHO-based approaches in extreme oligozoospermic and azoospermic men in an IVF setting. Results indicate that approximately one in four men classified as azoospermic by conventional semen analysis may actually have sperm present. The improved sensitivity of the SSSA technique may be of significant benefit to patients, particularly in fertility and assisted reproductive technique decision making.

Normal live birth after testicular sperm extraction and intracytoplasmic sperm injection in variant primary ciliary dyskinesia with completely immotile sperm and structurally abnormal sperm tails.

OBJECTIVE: To report on the investigation and fertility management of variant primary ciliary dyskinesia (PCD). DESIGN: Case report. SETTING: University-affiliated assisted reproductive technologies practice. PATIENT(S): A 40 year-old man presenting with 12 months' primary infertility, complete sperm immotility, severe morphologic defects, and moderate sinopulmonary disease. INTERVENTION(S): Electron microscopy (EM) of sperm, nasal cilial function studies, open testis biopsy, and sperm extraction for intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Outcome of ICSI treatment using immotile testicular sperm. RESULT(S): EM revealed abnormal connecting pieces, shortened midpieces with attenuated mitochondrial sheaths, poorly developed annulus, abnormal outer dense fibers, and axonemes missing the two central mircotubules. Nasal ciliary beat frequency was subnormal and dyssynchronous. Immotile testicular sperm were selected for ICSI based on physical characteristics and fertilized 12 of 18 eggs. A single day-5 blastocyst achieved a normal pregnancy and delivery of a healthy 3,840-g girl at 38 weeks' gestation. CONCLUSION(S): Nonclassic PCD may present with structurally abnormal completely immotile sperm, with seemingly little prospect of fertility, and moderate respiratory dysfunction supporting the presence of an underlying ciliopathy. Despite testicular sperm also being immotile and showing profound structural defects that would seem to preclude fertilization, more morphologically normal sperm are capable of establishing a normal pregnancy.

Human embryo culture and assessment for the derivation of embryonic stem cells (ESC).

The culture and critical assessment of early human embryos during the first week of human development are reviewed for the derivation of ESC. Both normal and abnormal features are assessed by phase contrast microscopy of whole embryos and in serial sections of fixed material by light and electron microscopy (TEM). Normal embryos follow a time table of development and have equal blastomeres with minimal fragmentation and nuclear defects. Abnormal embryos show more fragmentation and nuclear aberrations such as micronucleation and multinucleation, reflected by aneuploidy, polyploidy, and mosaicism. The selection of normal embryos and the hardiest of embryos that survive to blastocysts is recommended for the derivation and culture of ESC.

In vitro fertilisation (IVF) with donor eggs in post-menopausal women: are there differences in pregnancy outcomes in women with premature ovarian failure (POF) compared with women with physiological age-related menopause?

PURPOSE: This study assessed pregnancy rates and obstetric outcomes in women with premature ovarian failure (Group A) with post-menopausal women > or =40 years (Group B) who had IVF +/- ICSI using donor eggs. METHODS: This was a retrospective analysis of 54 recipients with either premature ovarian failure or physiological menopause undergoing oocyte donation between 2000 and 2007 at Monash IVF. RESULTS: The average number of stimulated cycles required for a woman in group A and B to deliver a baby was 1.75 and 1.4 respectively. Both groups had high cumulative pregnancy rates; however, there was a statistically significant difference with regards to rates of complications. CONCLUSION: Oocyte donation in both premature ovarian failure and physiological menopause is highly successful and cumulative pregnancy rate is an important statistic which can be used to inform women seeking this technique. High rates of complications, in conjunction with individual risk-factor analysis needs to be considered when counselling post-menopausal women about oocyte donation.

Blastocyst transfer does not cause a sex-ratio imbalance.

OBJECTIVE: To determine whether either single or double fresh blastocyst transfers result in a sex-ratio imbalance in resulting offspring compared with transfers on day 2 or 3 and whether there is a correlation between rate of embryo development and sex of the embryo. DESIGN: Retrospective analysis. SETTING: Large IVF center. PATIENT(S): Four hundred thirty-five live births from single fresh ETs for the period January 2005 through July 2007 and 2,043 live births from double ETs for the same period. INTERVENTION(S): Statistical analysis performed on sex ratio of offspring resulting from transfers (day 2, day 3, day 4, and blastocyst), as well as on the stage of development reached for each day in culture analyzed on sex of the embryo. MAIN OUTCOME MEASURE(S): Sex ratio of offspring by day of transfer. Stage of embryonic development by sex for each day in culture. RESULT(S): There was no difference in sex ratio with blastocyst transfer (single or double). There was no difference in speed of embryonic development at any stage in vitro. CONCLUSION(S): Male embryos do not grow faster than female embryos in culture. Blastocyst transfer does not result in a sex-ratio imbalance in resulting offspring.

Is there an ideal stimulation regimen for IVF for poor responders and does it change with age?

PURPOSE: To determine whether there is a superior treatment modality for 'poor' responders. METHOD: Retrospective analysis of three stimulation regimens, with patients stratified based on age, stimulation regime and response in previous cycles ("poor' responder or "non poor" responder). Fertilisation, embryo utilisation and clinical pregnancy rates were assessed. There were a total of 1,608 cycles in the 'poor' responder and 8,489 cycles in the 'non poor' responder groups. RESULTS: In 'poor' responders there was no significant difference in fertilisation rate, nor utilisation rate between the three stimulation regimes and no differences in the pregnancy rate/initiated cycle irrespective of age and stimulation regimen in any of the groups. 'Non poor' responders had a significantly greater pregnancy rate/initiated cycle for all stimulation regimens in both age groups compared with 'poor' responders. CONCLUSION: This large retrospective study of 'poor' responders has not shown a difference in pregnancy rates/initiated cycle between stimulation regimens.