STUDY QUESTION Is the metabolic health of men conceived using ICSI different to that of IVF and spontaneously conceived (SC) men? SUMMARY ANSWER ICSI-conceived men aged 18-24 years, compared with SC controls, showed differences in some metabolic parameters including higher resting diastolic blood pressure (BP) and homeostasis model assessment for insulin resistance (HOMA-IR) scores, although the metabolic parameters of ICSI- and IVF-conceived singleton men were more comparable. WHAT IS KNOWN ALREADY Some studies suggest that IVF-conceived offspring may have poorer cardiovascular and metabolic profiles than SC children. Few studies have examined the metabolic health of ICSI-conceived offspring. STUDY DESIGN, SIZE, DURATION This cohort study compared the metabolic health of ICSI-conceived men to IVF-conceived and SC controls who were derived from prior cohorts. Participants included 121 ICSI-conceived men (including 100 singletons), 74 IVF-conceived controls (all singletons) and 688 SC controls (including 662 singletons). PARTICIPANTS/MATERIALS, SETTING, METHODS Resting systolic and diastolic BP (measured using an automated sphygmomanometer), height, weight, BMI, body surface area and fasting serum metabolic markers including fasting insulin, glucose, total cholesterol, high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol, triglycerides, highly sensitive C-reactive protein (hsCRP) and HOMA-IR were compared between groups. Data were analysed using multivariable linear regression adjusted for various covariates including age and education level. MAIN RESULTS AND THE ROLE OF CHANCE After adjusting for covariates, compared to 688 SC controls, 121 ICSI-conceived men had higher diastolic BP (β 4.9, 95% CI 1.1-8.7), lower fasting glucose (β -0.7, 95% CI -0.9 to -0.5), higher fasting insulin (ratio 2.2, 95% CI 1.6-3.0), higher HOMA-IR (ratio 1.9, 95% CI 1.4-2.6), higher HDLC (β 0.2, 95% CI 0.07-0.3) and lower hsCRP (ratio 0.4, 95% CI 0.2-0.7) levels. Compared to 74 IVF-conceived singletons, only glucose differed in the ICSI-conceived singleton men (β -0.4, 95% CI -0.7 to -0.1). No differences were seen in the paternal infertility subgroups. LIMITATIONS, REASONS FOR CAUTION The recruitment rate of ICSI-conceived men in this study was low and potential for recruitment bias exists. The ICSI-conceived men, the IVF-conceived men and SC controls were from different cohorts with different birth years and different geographical locations. Assessment of study groups and controls was not contemporaneous, and the measurements differed for some outcomes (BP, insulin, glucose, lipids and hsCRP). WIDER IMPLICATIONS OF THE FINDINGS These observations require confirmation in a larger study with a focus on potential mechanisms. Further efforts to identify whether health differences are due to parental characteristics and/or factors related to the ICSI procedure are also necessary. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by an Australian National Health and Medical Research Council Partnership Grant (NHMRC APP1140706) and was partially funded by the Monash IVF Research and Education Foundation. S.R.C. was supported through an Australian Government Research Training Program Scholarship. R.J.H. is supported by an NHMRC project grant (634457), and J.H. and R.I.M. have been supported by the NHMRC as Senior and Principal Research Fellows respectively (J.H. fellowship number: 1021252; R.I.M. fellowship number: 1022327). L.R. is a minority shareholder and the Group Medical Director for Monash IVF Group, and reports personal fees from Monash IVF Group and Ferring Australia, honoraria from Ferring Australia and travel fees from Merck Serono and MSD and Guerbet; R.J.H. is the Medical Director of Fertility Specialists of Western Australia and has equity in Western IVF; R.I.M. is a consultant for and shareholder of Monash IVF Group and S.R.C. reports personal fees from Besins Healthcare and nonfinancial support from Merck outside of the submitted work. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.

STUDY QUESTIONS: What are the long-term health and reproductive outcomes for young men conceived using ICSI whose fathers had spermatogenic failure (STF)? Are there epigenetic consequences of ICSI conception? WHAT IS KNOWN ALREADY: Currently, little is known about the health of ICSI-conceived adults, and in particular the health and reproductive potential of ICSI-conceived men whose fathers had STF. Only one group to date has assessed semen parameters and reproductive hormones in ICSI-conceived men and suggested higher rates of impaired semen quality compared to spontaneously conceived (SC) peers. Metabolic parameters in this same cohort of men were mostly comparable. No study has yet evaluated other aspects of adult health. STUDY DESIGN, SIZE, DURATION: This cohort study aims to evaluate the general health and development (aim 1), fertility and metabolic parameters (aim 2) and epigenetic signatures (aim 3) of ICSI-conceived sons whose fathers had STF (ICSI study group). There are three age-matched control groups: ICSI-conceived sons whose fathers had obstructive azoospermia (OAZ) and who will be recruited in this study, as well as IVF sons and SC sons, recruited from other studies. Of 1112 ICSI parents including fathers with STF and OAZ, 78% (n = 867) of mothers and 74% (n = 823) of fathers were traced and contacted. Recruitment of ICSI sons started in March 2017 and will finish in July 2020. Based on preliminary participation rates, we estimate the following sample size will be achieved for the ICSI study group: mothers n = 275, fathers n = 225, sons n = 115. Per aim, the sample sizes of OAZ-ICSI (estimated), IVF and SC controls are: Aim 1-OAZ-ICSI: 28 (maternal surveys)/12 (son surveys), IVF: 352 (maternal surveys)/244 (son surveys), SC: 428 (maternal surveys)/255 (son surveys); Aim 2-OAZ-ICSI: 12, IVF: 72 (metabolic data), SC: 391 (metabolic data)/365 (reproductive data); Aim 3-OAZ-ICSI: 12, IVF: 71, SC: 292. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eligible parents are those who underwent ICSI at one of two major infertility treatment centres in Victoria, Australia and gave birth to one or more males between January 1994 and January 2000. Eligible sons are those aged 18 years or older, whose fathers had STF or OAZ, and whose parents allow researchers to approach sons. IVF and SC controls are age-matched men derived from previous studies, some from the same source population. Participating ICSI parents and sons complete a questionnaire, the latter also undergoing a clinical assessment. Outcome measures include validated survey questions, physical examination (testicular volumes, BMI and resting blood pressure), reproductive hormones (testosterone, sex hormone-binding globulin, FSH, LH), serum metabolic parameters (fasting glucose, insulin, lipid profile, highly sensitive C-reactive protein) and semen analysis. For epigenetic and future genetic analyses, ICSI sons provide specimens of blood, saliva, sperm and seminal fluid while their parents provide a saliva sample. The primary outcomes of interest are the number of mother-reported hospitalisations of the son; son-reported quality of life; prevalence of moderate-severe oligozoospermia (sperm concentration <5 million/ml) and DNA methylation profile. For each outcome, differences between the ICSI study group and each control group will be investigated using multivariable linear and logistic regression for continuous and binary outcomes, respectively. Results will be presented as adjusted odds ratios and 95% CIs.

STUDY QUESTION: Are uterine natural killer (uNK) cell numbers and their distribution relative to endometrial arterioles altered in women with recurrent implantation failure (RIF) compared to women with embryo implantation success (IS)? SUMMARY ANSWER: uNK cell numbers and their distribution relative to endometrial arterioles are not significantly different in women with RIF compared to women in whom embryo implantation occurs SUCCESSFULLY FOLLOWING IVF. WHAT IS ALREADY KNOWN: uNK cells are regulators of decidual angiogenesis and spiral arteriole remodelling during early pregnancy. Although some studies have shown that uNK cell numbers may be altered in women with RIF, the methods used to measure uNK cell numbers have proven inconsistent, making reproduction of these results difficult. It is unclear, therefore, whether the results reported so far are reproducible. Moreover, it is not known how uNK cell numbers may impact IVF outcomes. Despite the lack of conclusive evidence, uNK cell numbers are often evaluated as a prognostic criterion in women undergoing assisted reproductive procedures. STUDY DESIGN, SIZE, DURATION: Endometrial pipelle biopsies were collected 6-8 days post-LH surge in natural cycles from women with RIF (n=14), women with IS (n=11) and women with potential RIF at the time of the study (PRIF; n=9) from 2013 to 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: uNK cells (i.e. CD56+ and/or CD16+ phenotypes) and their distribution relative to endometrial arterioles were investigated by standard immunohistochemistry protocols and quantified using Aperio ScanScopeXT images digitized by ImageJ and deconvoluted into binary images for single cell quantification using a Gaussian Blur and Yen algorithm. MAIN RESULTS AND THE ROLE OF CHANCE: There was no significant difference in the cell density of CD56+ or CD16+ uNK cells in women with RIF compared to women with IS or PRIF. There was a higher proportion of uNK cells in the distal regions compared to the regions closest to the arterioles in all patient groups. Further, we identified a significant reduction in uNK cell density in women who had a previous pregnancy compared to those who had not, regardless of their current implantation status. LARGE SCALE DATA: ot applicable. LIMITATIONS, REASONS FOR CAUTION: piral arterioles could not always be accurately identified by digital image analysis; therefore, all endometrial arterioles were selected and analysed. Patient numbers for the study were low. However, as the clinical phenotypes of each patient were well defined, and endometrial dating was accurately determined by three independent pathologists, differences between patient groups with respect to the uNK numbers and distribution should have been measurable if uNK cell counts were to be useful as a prognostic marker of RIF. WIDER IMPLICATIONS OF THE FINDINGS: ur findings demonstrate that CD56+ and CD16+ uNK cell numbers are not significantly different in women with RIF in a typical cohort of women undergoing IVF. Further, prior pregnancy was associated with a significantly reduced number of uNK cells in both the RIF and IS patient groups, suggestive of a long-term pregnancy induced suppression of uNK cells. Combined, these findings do not support the clinical value of using uNK cell numbers as a prognostic indicator of implantation success with IVF treatment.

It may be assumed that infertility is not a problem in resource-poor areas where fertility rates are high. However, evidence overwhelmingly shows that childlessness is highly stigmatized in these settings and that women who are unable to bear children suffer significant social and psychological consequences. The World Health Organization has recommended that infertility be considered a global health problem and stated the need for ART to be adapted to low-resource settings. This paper describes a model for improving access to ART in low-resource settings. Experienced ART health professionals from Australia and Italy representing medical science, embryology, nursing and counselling used knowledge transfer to support a clinician, a laboratory scientist and a nurse to establish an ART service in Harare, Zimbabwe. Support and mentorship provided between October 2016 and December 2017 included: hosting the clinician and the embryologist for the new service in established ART clinics for short periods and providing them with dedicated mentorship and training during their stay; funding an experienced embryologist to travel to Zimbabwe (three times) to oversee the setting up of the lab and provide hands-on embryology training; funding a scientist and a nurse to travel to Zimbabwe to troubleshoot and establish protocols for record keeping and psychosocial care; and contributing approximately AUD $15,000 to the purchase of some equipment. By 31 March 2018, the team at IVF Zimbabwe had performed 166 ART procedures, which at time of writing had resulted in 16 births and 4 ongoing pregnancies. This case study demonstrates that with mentorship and modest financial support from ART experts from high-income settings, health professionals in low-income settings can deliver affordable ART with successful outcomes.

Despite the recent increase in human ovarian tissue banking, there has been little progress in establishing whether follicles within this tissue are viable and capable of function following cryopreservation. Two methods to assess growth and developmental potential of cryopreserved tissue are evaluated; (1) isolated follicle culture and (2) xenografting of tissue into a host animal. Development of numerous antral follicles following xenografting of cryopreserved tissue indicates that the cryopreservation procedure can preserve the developmental competence of primordial follicles.

BACKGROUND:In all IVF programmes, some patients fail to achieve an ongoing pregnancy, even after numerous embryo transfer procedures. An unfavourable environment within the uterus might be a contributory factor to such recurrent implantation failure. This question was addressed by measuring cytokine concentrations and matrix metalloproteinase activities in fluid derived from uterine irrigation of such patients.METHODS AND RESULTS:The uterine cavities of 22 patients who had previously undergone embryo transfer of at least 10 embryos without ongoing pregnancy were irrigated during the luteal phase. The resultant fluid was assayed for the concentration of interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, leukaemia inhibitory factor (LIF), IL-10 and matrix metalloproteinase (MMP) -2 and -9 activity. The results were compared with those of a control population of women known to be previously fertile (n = 16) and also with women with recurrent spontaneous abortion (n = 13). In the recurrent implantation failure group, the MMP score and IL-1beta concentration were significantly higher than those in the control group, whereas concentrations of IFN-gamma and IL-10 were significantly lower.CONCLUSIONS:In IVF patients with recurrent implantation failure, an altered pattern of intra-uterine cytokine concentration and MMP activity was observed.

Introduction: Recent studies have established that growth and development of follicles to the antral stage is possible within cryopreserved human ovarian tissue following autologous and heterologous transplantation. Although cryopreservation appears to have preserved the capacity for follicular development to the antral stage, evidence of ovulation is limited to indirect observations of a single follicle while the majority of antral follicles appear to be destined for atresia. The aim of this study was to assess the response of antral follicles within xenografts of cryopreserved ovarian tissue to administration of exogeneous hCG. Methods: Ovarian tissue was donated from 2 patients at risk of loss of fertility. Thin slices of ovarian cortex were cryopreserved as previously described. Fresh and cryopreserved tissue pieces were transplanted into female immunodeficient (SCID) mice > 20 weeks old. A single piece (0.5 x 1 x 1mm) of ovarian cortex was placed under the capsule of each kidney and mice were oophorectomised bilaterally. Intraperitoneal injections of gonadotrophin (1 IU/ml rec FSH) or saline were given on every second day. At ≥ 36 weeks post transplantation mice received an intraperitoneal injection of 20 IU of hCG. Kidneys were removed 15 and 30 hours after hCG, examined for gross morphological changes in the antral follicular appearance and fixed for histology. Results: The frequency and size of antral follicles observed were consistent with our previous results. No obvious difference was observed in gross morphology or histology between fresh or frozen tissue following transplantation or with administration of gonadotropin. On examination, the transition from a transparent to an opaque antral cavity was apparent 15 hours after hCG in all follicles. In these follicles, histological examination identified only minor changes consisting of loss of continuity in the inner layers of mural granulosa cells which slough into the cavity, and the presence of an occasional luteinized granulosa cell situated adjacent to these sloughing granulosa cells. Low numbers of luteinized cells were also observed amongst the stromal cells of the ovarian cortex. In contrast, histological examination of follicles fixed 30 hours after hCG revealed large areas of ovarian cortex, in close prioximity to the antral follicles, which contained luteinized cells. Luteinized cells and red blood cells were now present within both the follicle wall and the antral cavity. The granulosa cell layers had lost their organisation and appeared to be reduced in numbers. Similarly, the cumulus cells were now interspersed by an amorphous matrix and the germinal vesicle was absent or undergoing breakdown within the oocyte. Conclusion: These observations indicate a responsiveness of the cryopreserved ovarian tissue and follicles to hCG, suggesting preservation of the ability to undergo periovulatory changes, under appropriate conditions.

Using comparative genomic hybridization, we have detected chromosome abnormality in 76/126 (60%) single blastomeres biopsied prior to implantation from embryos from 20 women with repeated implantation failure following IVF. The abnormalities detected included aneuploidy for one or two chromosomes [32/126 (25%)] and complex chromosomal abnormality [37/126 (29%)]. Most of the chromosomes involved in single aneuploidy were those commonly found in live births or spontaneously aborted fetuses, whereas a greater range of chromosomes were involved in double aneuploidy. In blastomeres with complex abnormality, random and extensive loss and gain of all the chromosomes was observed. Further blastomeres from 25 embryos with single or double aneuploidy and 11 embryos with complex abnormality were analysed following embryo disaggregation. The specific abnormality was confirmed in the majority of cases and in some cases could be assigned as errors in meiotic or mitotic segregation. Complex abnormalities, suggestive of errors in cell cycle regulation, were present in a slightly higher proportion of these embryos than were seen in our previously studied cohort of surplus embryos. The disruption of the normal sequence of chromosome replication and segregation in early human embryos, caused either by maternal cytoplasmic factors or mutations in cell cycle control genes, may be a common cause of repeated implantation failure.

Embryos that are transferred after in vitro fertilization frequently fail to implant in the uterus; in many cases, this failure is due to chromosomal abnormalities in the embryo. The current methods of diagnosis of aneuploidy cannot detect many chromosomal abnormalities. This report on the case of a 38-year-old woman with a long history of infertility describes the use of comparative genomic hybridization to determine that all the chromosomes of a single embryo were free of aneuploidy, resulting in the birth of a healthy infant.

Human embryos have a high frequency of aneuploidy. Fluorescent in situ hybridization (FISH) on biopsied blastomeres has been used to select embryos for transfer to patients who have recurrent implantation failures (RIF) after IVF. However, some aneuploid embryos are diagnosed as “normal” because FISH provides an incomplete karyotype. Comparative genomic hybridization (CGH) is a molecular cytogenetic technique that screens the entire genome for aneuploidy. We have developed single cell CGH that identifies aneuploidy in fibroblasts (1Voullaire L. Wilton L. Slater H. Williamson R. Prenat Diagn. 1999; 19: 846-851Crossref PubMed Scopus (106) Google Scholar) and blastomeres (2Voullaire L. Williamson R. Slater H. Wilton L. Hum Genet. 2000; 106: 210-217Crossref PubMed Scopus (288) Google Scholar). We have now applied CGH to embryos from patients with RIF to select chromosomally normal embryos for transfer. For CGH, the whole genome of each blastomere was amplified using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). The blastomere (test) DNA was labelled green and normal genomic (reference) DNA was labelled red and hybridized to normal metaphase slides. The average green:red fluorescence ratio (G:R) for each chromosome was used to detect aneuploidy. When the DNA copy number is greater in the test sample the G:R is >1; when it is less in the test sample the G:R is <1. Fourteen embryos that were no longer required by patients were disaggregated into 77 single blastomeres and subjected to CGH (2Voullaire L. Williamson R. Slater H. Wilton L. Hum Genet. 2000; 106: 210-217Crossref PubMed Scopus (288) Google Scholar). CGH was successful in 75 (97%) blastomeres. CGH showed that only three (21%) embryos were chromosomally normal in every cell. Other abnormalities included monosomies, extensive aneuploidy and mosaicism. Nine couples with RIF had CGH applied to single cells biopsied from some or all of their embryos. CGH takes 4–5 days and so embryos were frozen after biopsy awaiting results. CGH was successful on 53/57 (93%) of embryos and 19/53 (36%) of embryos were normal for all chromosomes. Interestingly, more than half of the embryos diagnosed as abnormal by CGH would have been diagnosed as normal using our practice of FISH for chromosomes 13, 16, 18, 21 and 22. This work represents the most extensive chromosomal screening of embryos to date and the first application of CGH to clinical preimplantation genetic diagnosis. An ongoing comparison of FISH and CGH will determine whether the complete karyotype provided by CGH will enable improved selection of diploid embryos for transfer in patients with RIF.

The incidence of chromosomal aneuploidy in human oocytes is higher than for various animal species. Since this estimate for aneuploidies is based on data obtained from in-vitro fertilization (IVF) patients, it is possible that superovulation could be contributing to this phenomenon. In this study we determine the incidence of chromosomal aneuploidy in nonstimulated uninseminated human oocytes donated by IVF patients. Furthermore, we compare this incidence of aneuploidy to that obtained after superovulation using two different protocols for induction of multiple follicular growth. The rate of aneuploidy in non-stimulated oocytes was 20% (4/20). This is not significantly different from the rate of aneuploidy in oocytes obtained after superovulation with clomiphene/human menopausal gonadotrophin (HMG)/(HCG) (15/43 = 35%, chi 2 = 1.11; P > 0.20), buserelin-flare (8/25 = 32%; chi 2 = 0.32; P > 0.05), and the rate of aneuploidy in the total number of superovulated oocytes (23/68 = 34%; chi 2 = 82; P < 0.30). Furthermore, the incidence of chromosome aneuploidy in non-stimulated uninseminated oocytes (20%) was well within the range and not significantly different from that reported in the literature for both superovulated uninseminated oocytes (range, 21-57%; total aneuploidy rate, 67/216 = 31%; P < 0.30) and superovulated inseminated oocytes (range, 3-56%; total aneuploidy, 339/1480 = 23%; P < 0.95). Consequently, the data provide evidence that superovulation protocols used in IVF may not be responsible for the higher rate of aneuploidy in human oocytes. These results are discussed in relation to hypotheses on the occurrence of meiotic non-disjunction.