Bisphenol A correlates with fewer retrieved oocytes in women with tubal factor infertility.

PURPOSE: Serum and urinary bisphenol A (BPA) concentrations have been associated negatively with the number of retrieved oocytes after in vitro fertilization (IVF). The impact of BPA upon women with polycystic ovary syndrome (PCOS) and women with tubal factor infertility (TFI), following IVF, was investigated. To this purpose, associations among serum and urinary and follicular fluid (FF) BPA concentrations and the number of retrieved and fertilized oocytes and comparisons between pregnancy rates were evaluated. METHODS: This was a cross-sectional study conducted at a university-affiliated assisted conception unit between January and November 2019, including 93 women of reproductive age (PCOS: 45; TFI: 48) following IVF. Unconjugated FF and serum BPA concentrations and total urinary BPA concentration were measured using a novel gas chromatography-mass spectrometry method. The number of retrieved and fertilized oocytes and pregnancy rate were documented and evaluated. RESULTS: The number of oocytes retrieved from PCOS women was greater than that of 21 TFI women, independently of BMI. Lower FF BPA concentrations were found in all PCOS women and in overweight/obese PCOS compared to TFI women (0.50, 0.38, and 1.13 ng/mL, respectively). In TFI women, FF BPA concentrations correlated negatively with the number of retrieved oocytes. Serum and FF and urinary BPA concentrations did not significantly affect the number of fertilized oocytes and pregnancy rate in both groups. CONCLUSION: FF BPA concentrations were lower in all PCOS women and in overweight/obese PCOS than in TFI women. In TFI women, FF BPA concentrations correlated negatively with retrieved oocytes. Confirmation of these findings might lead to moderation of use of BPA-containing products by women undergoing IVF.

Adherence to the Mediterranean diet and IVF success rate among non-obese women attempting fertility.

STUDY QUESTION: Is adherence to the Mediterranean diet (MedDiet) associated with better IVF performance in women attempting fertility? SUMMARY ANSWER: Greater adherence to the MedDiet, defined using the validated Mediterranean diet score (MedDietScore), was associated with a higher likelihood of achieving clinical pregnancy and live birth among non-obese women <35 years of age. WHAT IS KNOWN ALREADY: Diet impacts fertility and certain nutrients and food groups appear to have a greater effect on reproductive health, but there are relatively few published data on the role of dietary patterns, and the MedDiet in particular, on assisted reproductive performance. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 244 non-obese women (22-41 years of age; BMI < 30 kg/m2) who underwent a first IVF treatment in an Assisted Conception Unit in Athens, Greece, between November 2013 and September 2016. The study was designed to evaluate the influence of habitual dietary intake and lifestyle on fertility outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Diet was assessed before the IVF treatment via a validated food-frequency questionnaire. Adherence to the MedDiet was assessed through the MedDietScore (range: 0-55), with higher scores indicating greater adherence. Intermediate outcomes (oocyte yield, fertilization rate and embryo quality measures) and clinical endpoints (implantation, clinical pregnancy and live birth) were abstracted from electronic medical records. Associations between MedDietScore and IVF outcomes were analysed using generalized linear models adjusting for age, ovarian stimulation protocol, BMI, physical activity, anxiety levels, infertility diagnosis, caloric intake and supplements use. MAIN RESULTS AND THE ROLE OF CHANCE: No association of MedDietScore with any of the intermediate outcomes or with implantation was found. However, compared with women in the highest tertile of the MedDietScore (≥36, n = 86), women in the lowest tertile (≤30, n = 79) had significantly lower rates of clinical pregnancy (29.1 vs 50.0%, P = 0.01) and live birth (26.6 vs 48.8%, P = 0.01). The multivariable-adjusted relative risk (95% CI) for clinical pregnancy comparing women in the lowest with women in the highest tertile of the MedDietScore was 0.35 (0.16-0.78; P-trend=0.01), and for live birth it was 0.32 (0.14-0.71; P-trend = 0.01). These associations were significantly modified by women's age (P-interaction <0.01 for both outcomes). MedDietScore was positively related to clinical pregnancy and live birth among women <35 years old (P ≤ 0.01) but not among women ≥35 years. Among women <35 years, a beneficial 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth. LIMITATIONS, REASONS FOR CAUTION: Our finding cannot be generalized to the whole reproductive population nor to obese women nor to women attending infertility clinics around the world. In addition, due to the observational study design, causal inference is limited. WIDER IMPLICATIONS OF THE FINDINGS: The results suggest that diet modifications and greater compliance to the Mediterranean diet may help increase the chances of a successful pregnancy and delivering a live baby for women undergoing IVF treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was partially supported by a grand from Harokopio University (KE321). All authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: NCT03050944.

The combination of calcium ionophore A23187 and GM-CSF can safely salvage aged human unfertilized oocytes after ICSI.

PURPOSE: Artificial oocyte activation using calcium ionophores and enhancement of embryonic developmental potential by the granulocyte-macrophage colony-stimulating factor (GM-CSF) have already been reported. In this study, we evaluated the synergistic effect of these two methods on aged human unfertilized oocytes after intracytoplasmic sperm injection (ICSI). Then, we cultured the resulting embryos to the blastocyst stage and screened them for chromosomal abnormalities, to assess the safety of this protocol. METHODS: Aged human oocytes deemed unfertilized after ICSI were activated, either by briefly applying the calcium ionophore A23187 alone (group A) or by briefly applying the ionophore and then supplementing the culture medium with recombinant human GM-CSF (rhGM-CSF) (group B). Next, the development was monitored in a time-lapse incubator system, and ploidy was analyzed by array comparative genomic hybridization (aCGH), after whole embryo biopsy and whole genome amplification. Differences between oocytes and resulting embryos in both groups were evaluated statistically. RESULTS: Oocytes unfertilized after ICSI can be activated with the calcium ionophore A23187 to show two pronuclei and two polar bodies. Addition of rhGM-CSF in the culture medium of A23187-activated oocytes enhances their cleaving and blastulation potential and results in more euploid blastocysts compared to the culture medium alone. CONCLUSIONS: This study shows that activating post-ICSI aged human unfertilized oocytes with a combination of a calcium ionophore and a cytokine can produce good-morphology euploid blastocysts.

Association between adherence to the Mediterranean diet and semen quality parameters in male partners of couples attempting fertility.

STUDY QUESTION: Is adherence to the Mediterranean diet (MedDiet) associated with better semen quality in men of subfertile couples attempting fertility? SUMMARY ANSWER: Greater adherence to the MedDiet, as assessed through the validated Mediterranean diet score (MedDietScore), was significantly associated with higher sperm concentration, total sperm count and sperm motility. WHAT IS KNOWN ALREADY: A-posteriori dietary pattern approaches have revealed that dietary patterns characterized by high intakes of fruits, vegetables, whole grains, fish and low intake of meat are associated with better semen quality. Yet, whether adherence to the MedDiet is associated with better semen profile remains largely unexplored. STUDY DESIGN, SIZE, AND DURATION: This was a cross-sectional study of 225 men from couples attending a fertility clinic in Athens, Greece, recruited between November 2013 and May 2016. The study was designed to evaluate the influence of habitual dietary intake and lifestyle on fertility outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Men aged 26-55 years, 51.1% overweight or obese, 20.9% smokers, with complete dietary data were analyzed. Diet was assessed via a food-frequency questionnaire and adherence to the MedDiet was assessed through the MedDietScore (range: 0-55; higher scores indicating greater adherence to MedDiet). Semen quality was evaluated according to World Health Organization 2010 guidelines. Multiple logistic regression analysis was used to evaluate associations between tertiles of the MedDietScore and the likelihood of having abnormal semen parameters, after adjusting for potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to men in the highest tertile of the MedDietScore (≥37, N = 66), a higher percentage of men in the lowest tertile of the score (≤30, N = 76) exhibited below the WHO reference values for sperm concentration (47.4% vs 16.7%, P < 0.001), total sperm count (55.3% vs 22.7%, P < 0.001), total motility (65.8% vs 31.8%, P < 0.001), progressive motility (84.2 vs 62.1%, P = 0.011) and sperm morphology (50.0 vs 28.8%, P = 0.023). In the multivariable adjusted models, men in the lowest tertile of the MedDietScore had ~2.6 times higher likelihood of having abnormal sperm concentration, total sperm count and motility, compared to men in the highest tertile of the score. LIMITATIONS, REASONS FOR CAUTION: The main limitation of the study stems from its cross-sectional nature, limiting our ability to determine causality. WIDER IMPLICATIONS OF THE FINDINGS: The results suggest that greater compliance to the MedDiet may help improve semen quality. Whether this translates into differences in male fertility remains to be elucidated. Our findings are consistent with previous studies showing that dietary patterns with some of the characteristics of the MedDiet, i.e. rich in fruit, vegetables, legumes and whole grains, are associated with better measures of semen quality. STUDY FUNDING/COMPETING INTERESTS: No funding was obtained. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: n/a.

Polar body analysis by array comparative genomic hybridization accurately predicts aneuploidies of maternal meiotic origin in cleavage stage embryos of women of advanced maternal age.

STUDY QUESTION: How accurate is array comparative genomic hybridization (array CGH) analysis of the first polar body (PB1) and second polar body (PB2) in predicting aneuploidies of maternal meiotic origin in the cleavage stage embryos of women of advanced maternal age? SUMMARY ANSWER: Almost all of the aneuploidies detected in cleavage stage embryos were associated with copy number changes in the polar bodies (93%) and all but one (98.5%) were predicted to be aneuploid. A minority of copy number changes (17%), mainly in PB1, did not result in the predicted changes in the embryo, but many of these were small copy number changes, which are likely to be artefacts. WHAT IS KNOWN ALREADY: Chromosome aneuploidy is a major cause of pregnancy failure and loss, abnormal pregnancy and live births. Most aneuploidy is of maternal meiotic origin and increases exponentially in the decade preceding the menopause. A pilot study demonstrated a high rate of concordance between the chromosomal status predicted by polar body analysis and the corresponding zygotes in women of advanced maternal age. STUDY DESIGN, SIZE AND DURATION: Polar body biopsy and array CGH analysis of mature oocytes, which fertilized normally, to identify segregation errors in meiosis, followed by the analysis of the corresponding cleavage stage embryos (n = 34), in a consecutive series of stimulated and natural IVF cycles in women of advanced maternal age. MATERIALS, SETTING AND METHODS: Twenty couples requesting aneuploidy screening (mean ± SD of maternal age 39 ± 3 years) had 16 controlled ovarian hyperstimulation and 7 natural IVF cycles. PB1 and PB2 were biopsied from mature oocytes, prior to intracytoplasmic sperm injection (ICSI) and following confirmation of normal fertilization, respectively. Array CGH was used to detect chromosome copy number changes and to predict aneuploidy in the corresponding embryos. Embryos with normal copy number in both polar bodies were transferred but, 34 cleavage stage embryos, most of which were predicted to have one or more aneuploidies of maternal meiotic origin, were analysed in whole after removal of the zona by array CGH, on Day 3 post-ICSI. MAIN RESULTS AND THE ROLE OF CHANCE: Thirty cleavage stage embryos, predicted to have one or more aneuploidies, were all confirmed to be aneuploid (100% concordant). Seventy four aneuploidies were detected in these embryos. Sixty-nine (93%) aneuploidies were associated with copy number changes in the polar bodies and 68 (98.5%) of these had been predicted to be aneuploid. Also, 19 of 20 (95%) balanced combinations of chromatid gain/loss in PB1/PB2 accurately predicted normal copy number in the corresponding embryos. However, 17 (12%) copy number changes in the polar bodies did not result in the expected outcome, including 12 false positive predictions of aneuploidy. Most of these involved copy number changes that were smaller than would be expected for whole chromosome or chromatid imbalance and occurred significantly more often in PB1 than PB2 (P < 0.0005). Three other embryos with only small copy number changes and one embryo with a partial chromosome loss in PB2, were all confirmed to be euploid. LIMITATIONS, REASONS FOR CAUTION: Accurate false positive and negative rates will require follow-up of both euploid and aneuploid embryos, ideally using molecular genetic markers to detect aneuploidy independently and to identify their origin. WIDER IMPLICATIONS OF THE FINDINGS: Polar body biopsy and array CGH analysis is efficient and accurately predicts most aneuploidies in cleavage stage embryos. However, the size of the ratio shifts, particularly in PB1, should always be compared with the X chromosome shift before it can be concluded that there is a real copy number change. STUDY FUNDING/COMPETING INTEREST(S): Study funded by Embryogenesis, Athens. P.S. and A.H.H. are employed full time and part time, respectively, by BlueGnome Ltd, Cambridge, UK.

Economic evaluation of alternative assisted reproduction techniques in management of infertility in Greece.

BACKGROUND: The purpose of this study was to compare Gonal-F(®), a recombinant follicle-stimulating hormone, with Menopur(®), a highly purified human menopausal gonadotrophin (hpHMG) in assisted reproduction in Greece. METHODS: A decision tree in combination with a Markov model was used to assess the clinical and economical impact of comparators for up to three consecutive cycles. Transition probabilities were derived from the literature and validated by clinical experts. Cost components were derived from the electronic databases of selected private and public clinics. A probabilistic sensitivity analysis was performed to deal with uncertainty and to construct a cost-effectiveness acceptability curve. RESULTS: There was a statistically significant difference in favor of the recombinant follicle-stimulating hormone arm compared with hpHMG, which was associated with 52 more births (95% uncertainty interval 26-78, P = 0.001) per 1000 patients. The cost per birth was estimated at €16,906 and €17,286 in the recombinant follicle-stimulating hormone and hpHMG arms, respectively. The cost per in vitro fertilization was estimated at €4365 in the recombinant follicle-stimulating hormone arm and €3815 in the hpHMG arm, indicating a difference of €550. The incremental cost per birth for recombinant follicle-stimulating hormone versus hpHMG was estimated at €14,540, while the incremental cost per life-year was estimated at €175.41. CONCLUSION: Recombinant follicle-stimulating hormone may represent a cost-effective choice compared with hpHMG when used for ovarian stimulation for a pharmacoeconomic point of view in the Greek public health care setting. However, it must be noted that in clinical practice both agents may be used together to increase the number of follicles, oocytes, embryos, and/or pregnancies in treated patients, an approach which has not been evaluated in Greece or reported in the literature due to obvious limitations.

Successful bone marrow transplantation in a pediatric patient with chronic myeloid leukemia from a HLA-identical sibling selected by preimplantation HLA testing.

We report successful bone marrow transplantation in an 11-year-old male with chronic myeloid leukemia from his HLA-identical sibling selected by preimplantation HLA testing. Because collection of cord blood failed, the transplantation was performed when the donor reached the age of 19 months, and sufficient bone marrow could be harvested safely. The patient was BCR/ABL negative at the time of transplantation after complete molecular response to imatinib. Currently, 16 months post-transplantation he is well and in complete molecular remission. This report describes preimplantation HLA-genotyping to deliver a matched sibling donor for successful transplantation of a malignant disorder.

Insulinlike growth factor-1Ec (MGF) expression in eutopic and ectopic endometrium: characterization of the MGF E-peptide actions in vitro.

The transcription of the insulinlike growth factor 1 (igf-1) gene generates three mRNA isoforms, namely IGF-1Ea, IGF-1Eb and IGF-1Ec (or MGF [mechano growth factor]). Herein, we analyzed the expression of IGF-1 isoforms in eutopic and ectopic endometrium (red lesions and endometriotic cysts) of women with endometriosis, and we characterized the actions of a synthetic MGF E-peptide on KLE cells. Our data documented that all three igf-1 gene transcripts are expressed in the stromal cells of the eutopic and ectopic endometrium; however, endometriotic cysts contained significantly lower IGF-1 isoform expression, both at the mRNA and protein level, as was shown using semiquantitative PCR and immunohistochemical methods. In addition, the glandular cells of the eutopic endometrium did not express any of the IGF-1 isoforms; however, the glandular cells of the ectopic endometrium (red lesions) did express the IGF-1Ec at mRNA and protein level. Furthermore, synthetic MGF E-peptide, which comprised the last 24 amino acids of the MGF, stimulated the growth of the KLE cells. Experimental silencing of the type 1 IGF receptor (IGF-1R) and insulin receptor expression of KLE cells (siRNA knock-out methods) did not alter the mitogenic action of the synthetic MGF E-peptide, revealing that MGF E-peptide stimulates the growth of KLE cells via an IGF-1R-independent and insulin receptor-independent mechanism. These data suggest that the IGF-1Ec transcript might generate, apart from mature IGF-1 peptide, another posttranslational bioactive product that may have an important role in endometriosis pathophysiology.

An evaluation of PGD in clinical genetic services through 3 years application for prevention of beta-thalassaemia major and sickle cell thalassaemia.

PGD represents an alternative within prenatal diagnosis services, which avoids terminating affected on-going pregnancies. In Greece, prevention programmes for haemoglobinopathies, including the option of prenatal diagnosis, are well established. Following optimization of a single-cell genotyping strategy (designed to be applicable for the majority of beta-thalassaemia major or sickle thalassaemia genotype interactions) along with close collaboration with an IVF unit, we integrated the option of PGD for at-risk couples with a problematic reproductive history. A total of 59 couples requesting PGD were counselled, of whom 41 initiated 63 PGD cycles. Following standard assisted reproduction treatment for oocyte retrieval, 20 cycles were cancelled (too few oocytes and/or poor quality embryos), but in 43 cycles single blastomeres were biopsied from 3 day embryos and genotyped (total 302). Diagnosis was achieved for 236 embryos, and 100 of 125 unaffected embryos were transferred. Sixteen pregnancies were established, although six were lost within the first trimester. Ten pregnancies underwent second trimester prenatal diagnosis, with nine pregnancies (13 babies: six singletons, two twins and one triplet) confirmed unaffected, although one singleton was a PGD misdiagnosis and terminated. The triplet pregnancy was selectively reduced to twins, and nine pregnancies went to term, with 12 healthy babies born. This report highlights advantages, limitations and approaches towards improvement when incorporating PGD within genetic services for a common recessive disease.