Beatrice Fuzzi

HLA-G expression in early embryos is a fundamental prerequisite for the obtainment of pregnancy.

Different mechanisms mediated by the expression of the HLA‐class Ib HLA‐G products are suggested to account for the induction of immune tolerance against the paternal antigens of the fetus during pregnancy. Soluble HLA‐G antigens, mainly produced by cytotrophoblast cells at the materno‐fetal interface and circulating in the body fluids, show a capacity analogous to that of membrane‐boundstructures to inhibit NK cells. In the present report we have investigated, using specific ELISA, the presence of sHLA‐G molecules in culture supernatants of early embryos obtained by in vitro fertilization (IVF) before transfer. The data obtained from the analysis of 285 supernatants corresponding to 101 IVF procedures (43 IVF, 58 intracytoplasmic sperm injection) identify two groups of patients on the basis of sHLA‐G antigen presence. No differences in clinical parameters were observed between the groups, but positive embryo implantations occurred only in women showing sHLA‐G molecules in culture supernatants (Fishers exact p value 2.56×10–3). The results obtained indicate that expression of HLA‐G products in embryo cells is a mandatory, but not sufficient, prerequisite for the development of pregnancy.

Non-classic sHLA class I in human oocyte culture medium.

Soluble human leukocyte antigen (sHLA) class I molecules have been described in all human fluids. These molecules play a significant role in immune function. sHLA has been shown to produce tolerance and to induce apoptosis in cytotoxic alloreactive T cells. They are also present in the supernatant of many cultured cells. Similarly, non-classic HLA class I antigens in soluble form are present in human fluids. Among these, HLA-G is the most important because of its location in fetal tissue that suggests maternal immunological tolerance of the fetal semiallograft. In our present study we show that using two monoclonal antibodies, w6/32 and TP25.99, in the enzyme-linked immunosorbent assay allows the detection of non-classic sHLA class I molecules in the medium from human embryo cultures. The sample were collected from oocytes cultures. Oocyte donors were 11 women attending the in vitro fertilization program. The results showed a significant association (chi2 = 9.66, p = 0.002) between sHLA antigens and the oocyte cleavage rate measured 48 h after fertilization.

Lack of Histocompatibility Leukocyte Antigen-G expression in early embryos is not related to germinal defects or impairment of interleukin-10 production by embryos

The expression of Histocompatibility Leukocyte Antigen (HLA)-G molecules is a mandatory prerequisite for the development of pregnancy but no hypotheses have yet been advanced regarding the lack of HLA-G modulation expression in a percentage of early embryos obtained by in vitro fertilization (IVF). One possible hypothetical model assumes that the absence of regulation of HLA-G or impaired interleukin (IL)-10 secretion could be related to germinal defects. We investigated the presence of soluble HLA-G antigens in supernatants of single embryo cultures from couples admitted to a second fertilization procedure; these couples showed a complete absence of HLA-G modulation in the first cycles embryo supernatants (0/31). The results obtained in the second IVF cycle showed embryo supernatants positive for HLA-G (14/40), suggesting that the previous lack of antigen modulation is independent of germinal defects. Furthermore, since it has been reported that oocytes and early embryos can secrete IL-10, an anti-inflammatory cytokine produced by type 2 helper T cells that induces upregulation of HLA-G expression in monocytes and trophoblasts, we investigated the levels of IL-10 and soluble HLA-G in 40 embryo culture supernatants from 21 IVF cycles. No associations were observed between the presence of IL-10 and the production and concentrations of soluble HLA-G, or between IL-10 levels and pregnancy outcome. These results indicate that the lack of HLA-G production in early embryos is not related to germinal defects or to impairment in embryo IL-10 secretion but could be ascribed to possible uncorrected fertilization processes.

Effect of dalteparin sodium administration on IVF outcome in non-thrombophilic young women: a pilot study

This study evaluated whether heparin administration could affect IVF outcome. A total of 172 women, aged <40years, without laboratory findings of thrombophilia and undergoing their first IVF cycle, were randomly allocated to treatment (n=86) and control (n=86) groups. Patients allocated to the treatment group received low-molecular-weight heparin dalteparin sodium 2500IU s.c. daily, in addition to routine luteal phase support, from oocyte retrieval up to the day of the pregnancy test or up to the ninth week of pregnancy in the cases of positive human chorionic gonadotrophin. From the day after the oocyte retrieval, all patients began standard supplementation with vaginal progesterone 200mg twice a day. At the sixth week of pregnancy, patients underwent an ultrasound scan to assess the number/viability of gestational sacs. Implantation rates were 15% and 12% in the dalteparin and control groups, respectively. The clinical pregnancy rates/embryo transfers were 26% (19/73) and 20% (16/80), in the dalteparin and control groups, respectively, with live birth rates/embryo transfer of 21% (15/73) and 16% (13/80). Despite the lack of statistical significance, the increase in pregnancies observed in the treatment group may be considered as an important clinical point in the optimization of IVF clinical outcome.

Physiology: In-vivo studies on ovarian insulin-like growth factor I concentrations in human preovulatory follicles and human ovarian circulation

In some recent hypotheses, the ovary has been indicated as a source of insulin-like growth factor (IGF)-I, with synthesis regulated from local steroidal and non-steroidal substances. We measured IGF-I concentrations in both serum and follicular fluid of women undergoing in-vitro fertilization (IVF) and embryo transfer, in both induced and spontaneous cycles. It was found that serum and follicular IGF-I concentrations were correlated with follicular morphology, oocyte maturity, steroid concentrations and clinical characteristics of IVF cycles. In addition, we measured IGF-I concentrations in both peripheral and ovarian circulation to gain further detailed information on the contribution of the ovary to IGF-I production. The results of our study support the hypothesis that follicular IGF-I is probably derived by diffusion from peripheral circulation and that local production appears unlikely.

Effects of low day 3 luteinizing hormone levels on in vitro fertilization treatment outcome

In a previous study we demonstrated that women with day 3 luteinizing hormone (LH) values < 3 IU/l subjected to controlled ovarian hyperstimulation without pituitary desensitization responded with a lower number of follicles > 15 mm compared to women with a higher basal LH level. The aim of this study was to determine whether in patients with day 3 LH levels < 3 IU/l a further reduction of serum LH concentration by gonadotropin-releasing hormone (GnRH) analog impairs follicular response to follicle stimulating hormone (FSH) and treatment outcome in in vitro fertilization (IVF) cycles. For this purpose we retrospectively studied 249 consecutive women subjected to standard IVF treatment employing pituitary desensitization with buserelin and follicular stimulation with urinary highly purified FSH. The patients were divided into two groups according to their day 3 LH value. The first group (group A) showed day 3 LH levels < 3 IU/l and the second (group B) had day 3 LH levels > 3 IU/l. Group A and B patients did not show statistically significant differences in the ovarian response to FSH, nor in IVF treatment outcome, showing that in FSH treated GnRH analog suppressed cycles, the ovarian responsiveness and IVF outcome do not differ according to basal LH values. However, the high dosage of FSH we employed in group A and B patients could account, at least in part, for this result. Indeed, comparative evaluations with unsuppressed cycles (our previous study) strongly suggest that a reduced ovarian responsiveness to gonadotropins in patients with day 3 LH values < 3 IU/l should be considered in clinical practice.