Luciana Criscuoli

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.

Hormonal patterns, steroid receptors and morphological pictures of endometrium in hyperstimulated IVF cycles

OBJECTIVE The purpose of this contribution is to investigate the pathophysiology of the abnormal endometrial development in hyperstimulated IVF cycles. STUDY DESIGN In 12 IVF-patients who did not have embryo transfer because of failure of oocyte fertilization, serum values of 17 beta-estradiol, progesterone, FSH, LH, total and free testosterone, and androstenedione were measured on the pick-up day and were evaluated with respect to the values normally expressed in the day of ovulation; in the endometrial specimens collected 2 days later, at the time of embryo replacement, estrogen and progesterone receptors were immunohistochemically determined and dating by the Noyes method was performed. RESULTS 17 beta-Estradiol values are constantly higher, and progesterone levels are, only in four cases, higher than expected for the day of ovulation in a natural cycle. These hormonal patterns can only partially explain the pattern of steroid receptors: progesterone receptors are expressed sparsely both in glands and stroma, while estrogen receptors are abundant in the glands and absent in the stroma. In 11 of 12 patients an abnormal endometrial development with stromal advancement was observed: this morphological picture of the endometrium could partially be explained only in the four cases presenting high progesterone levels by serum values and endometrial receptor content of estrogen and progesterone. CONCLUSIONS The abnormal endometrial development in hyperstimulated IVF cycles could only in part be explained by estrogen and progesterone, and other factors have to be considered.

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.

High variability in results of semen analysis in andrology laboratories in Tuscany (Italy): the experience of an external quality control (EQC) programme

We report the results of the first three trials of an external quality control (EQC) programme performed in 71 laboratories executing semen analysis in Tuscany Region (Italy). At the end of the second trial, participants were invited to attend a teaching course illustrating and inviting to adhere to procedures recommended by WHO (V edition). Results of the first three trials of the EQC documented a huge variability in the procedures and the results. The highest variability was found for morphology (CV above 80% for all the trials), followed by count (CV of about 60% for all the trials) and motility (CV below 30% for all the trials). When results of sperm count and morphology were divided according to the used method, mean CV values did not show significant differences. CV for morphology dropped significantly at the third trial for most methods, indicating the usefulness of the teaching course for morphology assessment. Conversely, no differences were observed after the course for motility and for most methods to evaluate count, although CV values were lower at the second and third trial for the laboratories using the Burker cytometer. When results were divided according to tertiles of activity, the lowest mean bias values (difference between each laboratory result and the median value of the results) for count and morphology were observed for laboratories in the third tertile (performing over 200 semen analysis/year). Of interest, mean bias values for concentration dropped significantly at the third trial for low activity laboratories. In conclusion, lack of agreement of results of semen analysis in Tuscany is mainly because of the activity and the experience of the laboratory. Our study points out the importance of participating in EQC programmes and periodical teaching courses as well as the use of WHO recommended standardized procedures to increase precision and to allow the use of WHO reference values.

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.

A Biochemical Approach to Detect Oxidative Stress in Infertile Women Undergoing Assisted Reproductive Technology Procedures

Oxidative stress plays a major role in critical biological processes in human reproduction. However, a reliable and biologically accurate indicator of this condition does not yet exist. On these bases, the aim of this study was to assess and compare the blood and follicular fluid (FF) redox status of 45 infertile subjects (and 45 age-matched controls) undergoing in vitro fertilization (IVF), and explore possible relationships between the assessed redox parameters and IVF outcomes. Reactive Oxygen Species (ROS) production, assessed by flow cytometry analysis in blood leukocytes and granulosa cells, significantly increased (p < 0.05) in infertile patients. Also, oxidative stress markers—ThioBarbituric Acid-Reactive Substances (TBARS) as an index of lipid peroxidation, and Oxygen Radical Absorbance Capacity (ORAC) to account for total antioxidant capacity, both assayed by fluorometric procedures—in blood and FF were significantly (p < 0.001) modified in infertile patients compared to the control group. Moreover, a significant correlation between blood redox markers and FF redox markers was evident. An ORAC/TBARS ratio, defined as the redox index (RI), was obtained in the plasma and FF of the patients and controls. In the patients, the plasma RI was about 3.4-fold (p < 0.0001) lower than the control, and the FF RI was about six-fold (p < 0.0001) lower than the control. Interestingly, both the plasma RI and FF RI results were significantly correlated (p < 0.05) to the considered outcome parameters (metaphase II, fertilization rate, and ongoing pregnancies). Given the reported findings, a strict monitoring of redox parameters in assisted reproductive techniques and infertility management is recommended.

Relevance of testicular histopathology on prediction of sperm retrieval rates in case of non-obstructive and obstructive azoospermia

Introduction: The aim of our research was to establish the relevance of testicular histopathology on sperm retrieval after testicular sperm extraction in patients with non-obstructive azoospermia and in patients with obstructive azoospermia, who already underwent a previous failure testicular fine needle aspiration. Methods: We evaluated a total of 82 azoospermic men, underwent testicular sperm extraction, referring to the Assisted Reproductive Technology Centre of the University of Florence, Italy between January 2008 and March 2017. A general and genital physical examination, scrotal and trans-rectal ultrasound, semen analysis, hormone measurements, including follicle-stimulating hormone, luteinizing hormone and total testosterone, were collected. Results: Successful sperm retrieval was obtained in 36 men of total (43.9%). Successful sperm retrieval was 29.5% in non-obstructive azoospermia patients, while men with obstructive azoospermia, who, underwent a previous failure testicular fine needle aspiration, had sperm retrieval in 86% of cases. Mean luteinizing hormone was 6.55 IU/L, total testosterone 4.70 ng/mL, right testicular volume 13.7 mL and left testicular volume 13.6 mL. Mean Follicle-stimulating hormone was 13.45 IU/L in patients with negative sperm retrieval and 8.18 IU/L in men with successful sperm retrieval. According to histology, 20.7% had normal spermatogenesis, 35.3% hypospermatogenesis, 35.3% maturation arrest and 8.5% Sertoli cell-only syndrome. Successful sperm retrieval was 88.2% in patients with normal spermatogenesis, 24.1% in the maturation arrest group and 48.27% in patients with hypospermatogenesis, while negative sperm retrieval was reported in Sertoli cell-only syndrome patients. Seven cases with maturation arrest showed a successful sperm retrieval. Conclusion: Testicular histopathology after testicular sperm extraction offers important information on prediction of sperm retrieval and can guide the surgeon in choosing the more suitable therapeutic practice.

Are hormone measurements and ultrasounds really predictors of sperm retrieval in testicular sperm extraction? A case report and literature review

Azoospermia can be diagnosed in about 10%–15% of the infertile male population. To overcome the problem of failure to produce spermatozoa in the ejaculate in patients with nonobstructive azoospermia (NOA), testicular sperm extraction (TESE) may be performed to find the focal area of spermatogenesis. A 47‐year‐old man with NOA presented for treatment of secondary couple infertility. The patient underwent a first TESE 7 years earlier with cryopreservation, and an intracytoplasmic sperm injection–embryo transfer ended in a term pregnancy. He reported a history of repeated testicular traumas. At the present time, a complete medical workup was carried out, including clinical history, general and genital physical examination, scrotal and transrectal ultrasounds. Hormone measurements showed follicle‐stimulating hormone level of 42.7 IU/L, luteinising hormone of 11.4 IU/L, total testosterone of 2.6 ng/ml and right and left testicular volume, respectively, of 4 and 3.9 ml. He underwent a second TESE, with successful sperm retrieval and cryopreservation. The histological pattern was hypospermatogenesis. In cases of extreme testicular impairment, although in the presence of very high follicle‐stimulating hormone value and small testicular volume, estimating poor sperm recovery potential, the integration of clinical and anamnestic data, could help the surgeon to practise the more appropriate method of treatment.