Ovarian follicular fluid levels of phthalates and benzophenones in relation to fertility outcomes.

BACKGROUND: Many endocrine disrupting chemicals (EDCs), for instance phthalates and benzophenones, are associated with adverse fertility outcomes and semen quality parameters. OBJECTIVE: To evaluate if concentrations of selected phthalate metabolites and benzophenones measured in follicular fluid are associated with fertility outcomes (i.e., reproductive hormones, antral follicle count, detected heartbeat at gestational week 7, and live birth) and, in a supplementary study, if measured concentrations of chemicals in follicular fluid can exert biological effects on human spermatozoa. METHODS: Overall, 111 couples from a fertility clinic in Denmark contributed with 155 follicular fluid samples. Concentrations of 43 metabolites from 19 phthalates and phthalate substitutes and six benzophenones were measured in follicular fluid using liquid chromatography-tandem mass spectrometry. Multiple linear and logistic regression with an applied generalized estimating equation model allowing more than one measurement per woman assessed the association between follicular EDC levels and fertility outcomes. The assessment of biological effects of individual and mixtures of EDCs on human spermatozoa was conducted through a human sperm cell based Ca2+-fluorimetric assay. RESULTS: Benzophenone-3 (BP-3) and seven metabolites of five phthalates were detectable in follicular fluid. Women with metabolites of dibutyl phthalate isomers in the highest tertiles had lower antral follicle count (MiBP: ß = -5.35 [95 % CI: -9.06; -2.00], MnBP: ß = -5.25 [95 % CI: -9.00; -2.00]) and lower odds for detecting a heartbeat at gestational week 7 (MiBP: OR = 0.35 [95 % CI: 0.14; 0.91], MnBP: OR = 0.39 [95 % CI: 0.13; 1.15]). Mixtures of the measured concentrations of BP-3 and the seven phthalate metabolites induced a small significant increase in the intracellular calcium ion concentration in human spermatozoa from healthy donors (n = 3). DISCUSSION: Phthalate metabolites and BP-3 were detectable in follicular fluid and high concentrations of some phthalate metabolites were linked with lower chance of successful fertility treatment outcomes. Chemical mixture concentrations in follicular fluid induced a calcium response in human spermatozoa highlighting possible biological effects at physiologically relevant concentrations.

Is paternal age associated with transfer day, developmental stage, morphology, and initial hCG-rise of the competent blastocyst leading to live birth? A multicenter cohort study.

In this study we investigated whether age of men undergoing assisted reproductive technology (ART) treatment was associated with day of transfer, stage, morphology, and initial hCG-rise of the competent blastocyst leading to a live birth? The design was a multicenter historical cohort study based on exposure (age) and outcome data (blastocyst stage and morphology and initial hCG-rise) from men whose partner underwent single blastocyst transfer resulting in singleton pregnancy/birth. The ART treatments were carried out at sixteen private and university-based public fertility clinics. We included 7246 men and women, who between 2014 and 2018 underwent controlled ovarian stimulation (COS) or Frozen-thawed Embryo Transfer (FET) with a single blastocyst transfer resulting in singleton pregnancy were identified. 4842 men with a partner giving birth were included, by linking data to the Danish Medical Birth Registry. We showed that the adjusted association between paternal age and transfer day in COS treatments was OR 1.06, 95% CI (1.00;1.13). Meaning that for every increase of one year, men had a 6% increased probability that the competent blastocyst was transferred on day 6 compared to day 5. Further we showed that the mean difference in hCG values when comparing paternal age group 30-34, 35-39 and 40-45 with the age group 25-29 in those receiving COS treatment, all showed significantly lower adjusted values for older men. In conclusion we hypothesize that the later transfer (day 6) in female partners of older men may be due to longer time spent by the oocyte to repair fragmented DNA of the sperm cells, which should be a focus of future research in men.

[Oocyt donation].

The number of treatments with donated oocytes has risen markedly in Denmark during the latest decade due to changes in legislation and because female age is increasingly advanced when fertility treatment is warranted. Today, oocyte donation is a standard procedure offering the otherwise untreatable a high chance of achieving a pregnancy. Live birth rates as high as 35% per treatment are being reported. It is, however, important to be aware of increased risks of hypertensive disorders and bleeding complications in these pregnancies. As pointed out in this review, continuous research in the field is therefore highly needed.

Association between women's age and stage, morphology, and implantation of the competent blastocyst: a multicenter cohort study.

OBJECTIVE: To study if the age of women undergoing assisted reproductive technology treatment associates with stage, morphology, and implantation of the competent blastocyst. DESIGN: Multicenter historical cohort study based on exposure (age) and outcome data (blastocyst stage and morphology and initial human chorionic gonadotrophin [hCG] rise) from women undergoing single blastocyst transfer resulting in singleton pregnancy/birth. SETTING: Sixteen private and university-based facilities. PATIENT(S): In this study, 7,246 women who, between 2014 and 2018, underwent controlled ovarian stimulation (COS) or frozen-thawed embryo transfer (FET) with a single blastocyst transfer resulting in singleton pregnancy were identified. Linking data to the Danish Medical Birth Registry resulted in a total of 4,842 women with a live birth being included. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The competent blastocyst development stage (1-6), inner cell mass (A, B, C), trophectoderm (A, B, C), and initial serum hCG value. RESULT(S): Adjusted analysis of age and stage in COS treatments showed that for every 1-year increase in age there was a 5% reduced probability of the competent blastocyst assessed as being in a high stage at transfer. Comparison between hCG values in women 18-24 years and 25-29 years in both COS and FET showed significantly lower levels in the youngest women. CONCLUSION(S): The initial hCG rise was influenced by the age of the woman, with an identical pattern for hCG values in COS and FET treatments. In COS, the competent blastocyst had a reduced stage with increasing women's age.

Presence of the vitamin D inactivating enzyme CYP24A1 in human sperm and prediction of the success of intrauterine insemination: A prospective study.

Male fertility is routinely evaluated by semen analysis, although semen quality variables such as; sperm count, motility and morphology have low predictive value for spontaneous pregnancies and fertility treatment outcomes. Vitamin D has been suggested to be beneficial for male reproduction. The vitamin D receptor and the vitamin D inactivating enzyme CYP24A1 are co-expressed in high quality sperm. Presence of CYP24A1 at the annulus of human sperm can distinguish between sperm from healthy and infertile men with high specificity and is positively correlated with semen quality. The high expression level in the testis of FAM57B2, which is activated by 24,25OH2D3, indicates an uncharacterized biological role for CYP24A1 in male reproduction. Moreover, activated vitamin D has been shown to induce sperm motility and promote fertilization in vitro. Here, we prospectively investigated whether the fraction of CYP24A1 positive sperm was a better predictor of clinical pregnancy than semen analysis by including 240 fertility treatments (169 couples) from a single fertility centre in Denmark. ROC-curve based analysis showed that the percentage of sperm expressing CYP24A1 was a better predictor of successful pregnancy outcome after intrauterine inseminations (IUI) than both sperm concentration and motility (p < 0.05). Interestingly, samples with CYP24A1 staining >67% of the sperm increased the likelihood of achieving pregnancy 4-fold after IUI compared with samples having fewer sperm with detectable CYP24A1 (p < 0.05). Neither CYP24A1 nor any of the other assessed semen quality variables were predictive for the treatment outcome of the more invasive assisted reproductive techniques (IVF and ICSI). In conclusion, our results provide proof of principle for a CYP24A1-based sperm test to improve fertility outcome for infertile patients referred for IUI and supports a role for vitamin D metabolites during fertilization.

Limitations of a time-lapse blastocyst prediction model: a large multicentre outcome analysis.

The goal of embryo selection models is to select embryos with the highest reproductive potential, whilst minimizing the rejection of viable embryos. Ultimately, any embryo selection model must be tested on clinical outcome. We therefore retrospectively tested a published blastocyst prediction model on a large combined set of transferred embryos with known clinical outcome. The model was somewhat effective in that we found a relative increase of 30% for implantation in the model-selected group of embryos. There was, however, a concomitant large rejection of embryos from our test cohort, which actually resulted in pregnancy. This hypothetical experiment highlights the limitations of predicting blastulation only. Crucially, it illustrates that both sensitivity and specificity are important parameters when developing embryo selection models for prospective clinical use.

Glycodelin in endometrial flushing fluid and endometrial biopsies from infertile and fertile women.

OBJECTIVE: To investigate in the natural cycle just before IVF, whether glycodelin levels in endometrial flushing fluid obtained days LH+1 and LH+7 can be used in predicting pregnancy in the following IVF cycle, and whether there are differences in women with tubal factor infertility compared to women with unexplained infertility and fertile controls. STUDY DESIGN: A prospective observational multicentre study of 21 fertile and 75 infertile women (25 showed abnormal tubes with no signs of hydrosalpinges, 18 had uni- or bi-lateral hydrosalpinges, 17 were salpingectomised because of hydrosalpinges, and 15 women had unexplained infertility). Endometrial flushing at days LH+1 and LH+7, endometrial biopsy, and blood sampling at day LH+7 were performed before down-regulation for IVF. Glycodelin levels in endometrial flushing fluids (EFF), biopsies, and plasma samples were related to tubal pathology, endometrial dating and IVF outcome. Furthermore, total protein concentration was measured in EFF to investigate the influence of normal endometrial maturation on protein concentrations from days LH+1 and LH+7. RESULTS: At day LH+1, EFF glycodelin levels were higher in infertile women with abnormal tubes compared to fertile women, particularly in women conceiving after the following IVF. For women with unexplained infertility, a higher level at day LH+1 was present only in women not conceiving after the following IVF. ROC curve analysis showed that at day LH+1 EFF glycodelin levels had no predictive value for IVF outcome. At day LH+7, glycodelin levels in endometrial flushing fluids and biopsies depended on endometrial dating. CONCLUSIONS: At day LH+1, glycodelin concentration is increased in endometrial flushing fluid from infertile women with abnormal tubes compared to fertile controls without being a valuable predictor of subsequent pregnancy. At day LH+7 the glycodelin level depends on endometrial dating.

Effect of levonorgestrel and mifepristone on endometrial receptivity markers in a three-dimensional human endometrial cell culture model.

OBJECTIVE: To investigate the effect of levonorgestrel and mifepristone on the expression of endometrial receptivity markers in a three-dimensional endometrial construct. DESIGN: In vitro study. SETTING: University hospital and research laboratory. PATIENT(S): Twelve fertile donors. INTERVENTION(S): Timed endometrial biopsy. MAIN OUTCOME MEASURE(S): Examine the effect of levonorgestrel along with another well-studied fertility-regulating drug, mifepristone, on the expression of endometrial receptivity factors in a three-dimensional stromal and epithelial cell coculture model by immunohistochemistry. RESULT(S): Both epithelial and stromal cells of in vitro endometrial construct showed the presence of estrogen receptor-alpha, estrogen receptor-beta, progesterone receptors-(A+B), vascular endothelial growth factor, leukemia inhibitory factor, interleukin-1 beta, and cyclooxygenase-2, whereas the expression of progesterone receptor-B (AR), integrin alpha(V)beta(3,) and MUC1 were confined to epithelial cells. Mifepristone up-regulated expression of epithelial estrogen receptor-beta and progesterone receptor-B and down-regulated stromal vascular endothelial growth factor and surface molecules MUC1 and integrin alpha(V)beta(3) as observed in vivo. Levonorgestrel had no effect on the expression of endometrial receptivity markers studied. CONCLUSION(S): This in vitro model expresses progesterone-regulated endometrial receptivity factors seen in the physiologic condition. Treatment with levonorgestrel did not affect the expression of these endometrial receptivity markers in contrast to mifepristone. This in vitro model holds the potential to study endometrial receptivity, the embryo-endometrial interaction, and develop new agents for fertility control.

Ultrasound characteristics and histological dating of the endometrium in a natural cycle in infertile women compared with fertile controls.

OBJECTIVE: To compare endometrial dating and ultrasound texture in a natural cycle before IVF and relate these to outcome and to fertile references. DESIGN: Prospective study with a fertile reference group. SETTING: Four university IVF clinics. PATIENT(S): Seventy-five IVF patients and 21 fertile women. INTERVENTION(S): Ultrasound, biopsy, blood sampling. In vitro fertilization-ET in the following cycle. MAIN OUTCOME MEASURE(S): Endometrial evaluation, P, IVF outcome. RESULT(S): At day LH+7, 42% infertile vs. 67% fertile women demonstrated endometria that were in phase (statistically nonsignificant difference). Nine percent had an accelerated endometrium, and 47% (infertile) vs. 24% (fertile) had a delayed endometrium (statistically nonsignificant difference). Statistically significantly fewer women with tubal factor and no hydrosalpinges had an endometrium in phase (20%) than was the case in fertile women (67%). Dating could not predict outcome. Statistically significantly more women in the hydrosalpinx group had a visible midline at day LH+7 compared with the case in other infertile groups. Forty-three percent conceived after IVF-ET. Plasma P was statistically significantly lower in nonpregnant women compared with in women with ongoing pregnancies and with fertile controls. A periovulatory hyperechogenic endometrium resulted in no ongoing pregnancies. CONCLUSION(S): In a natural cycle preceding IVF, a low midluteal P level predicts a low implantation rate. A periovulatory hyperechogenic endometrium or hydrosalpinges visible at ultrasound may have some predictive value. Endometrial dating was of no help.

The antiprogesterone Org 31710 inhibits human blastocyst-endometrial interactions in vitro.

OBJECTIVE: To investigate the effect of the anti-P Org 31710 on human blastocyst attachment to cultured endometrial epithelial cells. DESIGN: Experimental in vitro study. SETTING: University hospital. PATIENT(S): Eleven fertile endometrial donors. INTERVENTION(S): Timed endometrial biopsy for cell cultures. MAIN OUTCOME MEASURE(S): Blastocyst attachment rate on endometrial cell cultures; secretion of glycodelin and leukemia inhibitory factor into the culture medium measured by RIA and ELISA techniques; and expression of progesterone receptors, interleukin-1 receptor type-1, and integrin subunit beta(3) on endometrial epithelial cells examined by immunohistochemistry. Endometrial pinopodes visualized by scanning electron microscopy. RESULT(S): Eleven of 16 human blastocysts attached to control cultures, whereas none of 10 blastocysts attached when Org 31710 was added to the culture medium (P=.0007). Immunohistochemical studies demonstrated no significant differences between groups. Biochemical analyses displayed a trend toward higher glycodelin secretions and, by scanning electron microscopy, a tendency toward less pinopode formation in the Org 31710 group, but the results did not reach statistical significance. The presence of swollen microvilli, precursors of endometrial pinopodes, was significantly reduced on cultures with Org 31710 (P=.03). CONCLUSION(S): The study presents a model for human blastocyst-endometrial interactions responding to an anti-P drug. The exact mechanism for the anti-attachment properties of Org 31710 on the cultured endometrial cells and the blastocysts needs further evaluations.

Biochemical evaluation of endometrial function at the time of implantation.

OBJECTIVE: To review the literature on various endometrial factors assumed to be of importance to implantation and to evaluate their potential clinical value in the assessment of endometrial function at the time of implantation in infertile women in natural and stimulated cycles. DESIGN: Literature review. RESULT(S): Cytokines such as leukemia inhibitory factor, colony-stimulating factor-1, and interleukin-1 have all been shown to play important roles in the cascade of events that leads to implantation. They participate in a synchronized cooperation between the endometrium and the preimplanting embryo under the influence of steroid hormones. The same applies to the integrin alpha(v)beta(3), glycodelin, and the polymorphic mucin 1. The usefulness of these factors to assess endometrial receptivity and to estimate the prognosis for pregnancy in natural and artificial cycles remains to be proven. CONCLUSION(S): The studies performed to date have mostly included only small groups of patients with a lack of fertile controls, and only a few prospective, controlled trials have been carried out. Therefore, definite conclusions about the clinical value of these factors in the assessment of endometrial function and prognosis for pregnancy after artificial reproductive therapy cannot be drawn at present. Further evaluation of their importance for and function during implantation is needed.