Salvatore Dessole

Cryoprotectant-Free Cryopreservation of Human Spermatozoa by Vitrification and Freezing in Vapor: Effect on Motility, DNA Integrity, and Fertilization Ability

Abstract Human spermatozoa can be successfully cryopreserved avoiding the use of cryoprotectants through vitrification at very high cooling rates (up to 7.2 × 105 °C/min). This is achieved by directly plunging a copper cryoloop loaded with a sperm suspension into liquid nitrogen. After storage, vitrified spermatozoa are instantly thawed by melting in an agitated, warm medium. The goal of the present study was to compare the quality of spermatozoa cryopreserved using this rapid vitrification method with that of spermatozoa cooled relatively slowly by preexposure of the loaded cryoloop to liquid nitrogen vapor (−160°C) with speed in the range 150–250°C/min) before immersion into liquid nitrogen. Both cooling modes led to comparable results in terms of the motility, fertilization ability, and DNA integrity of the warmed spermatozoa. In both cases, instant thawing by melting in a warm medium was essential for successful cryopreservation. Our findings suggest that optimal regimes for the cryoprotectant-free cryopreservation of spermatozoa need not be restricted to very fast cooling before storage in liquid nitrogen, a wide range of cooling rates being acceptable. Herein, we discuss the implications of this finding in the light of the physics of extra- and intracellular vitrification.

Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success

The use of cryoprotective agents for the conventional cryopreservation of human spermatozoa, oocytes, zygotes, early cleavage stage embryos and blastocysts is an integral part of almost every human IVF programme. Moreover, the cryopreservation of these types of cells by direct plunging into liquid nitrogen usually requires high cryoprotectant concentrations with consequent cytotoxic effects. This review covers the history of this problem, and in this light offers an explanation, through physico-chemical concepts, for one of the most recent developments in this area: the recovery of motile and potent spermatozoa after cryoprotectant-free vitrification.

Lipoprotein metabolism during normal pregnancy

OBJECTIVE We sought to investigate the changes in circulating serum lipids and lipoproteins, including lipoprotein (a), and low-density lipoprotein size in women during normal pregnancy. STUDY DESIGN Twenty-two women (mean age, 31 +/- 5 years; 13 primiparous subjects) were studied during uncomplicated pregnancy with normal outcome. Twenty-four nulliparous women of similar age (31 +/- 4 years) were studied as control subjects. RESULTS Serum triglycerides and total and low-density lipoprotein cholesterol increased significantly during pregnancy in all women. Women with changes in low-density lipoprotein during the second and third trimesters showed a more marked increase in serum triglycerides, and this effect was slightly more evident in the multiparous subjects. No other differences were evident between primiparous and multiparous women apart from high-density lipoprotein cholesterol levels, which were slightly decreased in the latter subjects. CONCLUSIONS Our results show that during normal pregnancy, the increase in plasma triglycerides may lead to the appearance of the atherogenic dense low-density lipoproteins in a subgroup of women. We suggest that the observed changes in low-density lipoprotein patterns during pregnancy might be used to identify those women who later in life will have these atherogenic small and dense low-density lipoproteins.

Sonovaginography is a new technique for assessing rectovaginal endometriosis

OBJECTIVE To evaluate the efficacy of a new technique, the sonovaginography, for the assessment of rectovaginal endometriosis. DESIGN Prospective study. SETTING University hospital. PATIENT(S) Forty-six women were scheduled for laparotomic or laparoscopic surgery because of rectovaginal endometriosis suspected on the basis of patient history and/or clinical examination. INTERVENTION(S) Before surgery, all the women underwent transvaginal ultrasonography and then sonovaginography. The latter is based on transvaginal ultrasonography combined with the introduction of saline solution to the vagina that creates an acoustic window between the transvaginal probe and the surrounding structures of the vagina. Ultrasound findings were compared with the results of surgical exploration and histological examination. MAIN OUTCOME MEASURE(S) We assessed the accuracy of transvaginal ultrasonography and of sonovaginography for the detection and the location and extension assessment of rectovaginal endometriotic lesions, as well as compared patient compliance between the procedures. RESULT(S) Sonovaginography diagnosed rectovaginal endometriosis more accurately than did transvaginal ultrasonography, with a sensitivity and specificity of 90.6% and 85.7%, respectively, whereas the transvaginal ultrasonography has shown a sensitivity and specificity of 43.7% and 50%, respectively. Patient discomfort did not differ significantly between the procedures. CONCLUSION(S) Sonovaginography is a reliable and simple method for the assessment of rectovaginal endometriosis and provides information on location, extension, and infiltration of the lesions, which are important factors in selecting the kind of surgery.

Clean technique for cryoprotectant-free vitrification of human spermatozoa

Human spermatozoa can be successfully cryopreserved without the use of cryoprotectants through vitrification at very high warming rates. This is achieved by plunging a small amount of frozen sperm suspension into a warming medium, or a large amount of sperm suspension into an agitated warming medium. The aim of the present study was to compare the motility of human spermatozoa cryopreserved using four different methodologies of cooling and warming: cryoloops, droplets, open-pulled straws and standard open straws. Evaluation of two parameters, motility and viability rate of spermatozoa, suggests that all four methods are suitable for use in assisted reproductive technology. However, only the use of open-pulled straws as well as standard open straws allows the isolation of spermatozoa from liquid nitrogen with low potential risk of microbial contamination during freezing and storage, and is thereby a clean method of vitrification.

Efficacy of low-dose intravaginal estriol on urogenital aging in postmenopausal women

Objective To assess the efficacy and safety of intravaginal estriol administration on urinary incontinence, urogenital atrophy, and recurrent urinary tract infections in postmenopausal women. Design Eighty-eight postmenopausal women with urogenital aging symptoms were enrolled in this prospective, randomized, placebo-controlled study. Participants were randomly divided into two groups, with each group consisting of 44 women. Women in the treatment group received intravaginal estriol ovules: 1 ovule (1 mg) once daily for 2 weeks and then 2 ovules once weekly for a total of 6 months as maintenance therapy. Women in the control group received inert placebo vaginal suppositories in a similar regimen. We evaluated urogenital symptomatology, urine cultures, colposcopic findings, urethral cytologic findings, urethral pressure profiles, and urethrocystometry before as well as after 6 months of treatment. Results After therapy, the symptoms and signs of urogenital atrophy significantly improved in the treatment group in comparison with the control group. Thirty (68%) of the treated participants, and only seven (16%) of the control participants registered a subjective improvement of their incontinence. In the treated participants, we observed significant improvements of colposcopic findings, and there were statistically significant increases in mean maximum urethral pressure, in mean urethral closure pressure as well as in the abdominal pressure transmission ratio to the proximal urethra. Urethrocystometry showed positive but not statistically significant modifications. Conclusions Our results show that intravaginal administration of estriol may represent a satisfactory therapeutic choice for those postmenopausal women with urogenital tract disturbances who have contraindications or refuse to undergo standard hormone therapy.

Comparison between transvaginal sonography, saline contrast sonovaginography and magnetic resonance imaging in the diagnosis of posterior deep infiltrating endometriosis

To compare clinical evaluation, transvaginal sonography (TVS), saline contrast sonovaginography (SCSV) and magnetic resonance imaging (MRI) in the diagnosis of posterior deep pelvic endometriosis (DPE).

Postpartum ovarian vein thrombosis: an unpredictable event: two case reports and review of the literature

Abstract. Ovarian vein thrombosis (OVT) is a rare but serious postpartum complication that, in most cases, occurs in the right ovarian vein. Certain diagnosis, following clinical suspect because of lower quadrant tenderness and fever that alone does not respond to adequate broad-spectrum antibiotics, is now based on computed tomography (CT), although other imaging techniques, such as color Doppler ultrasonography and magnetic resonance (MR) imaging, are useful. Heparin and intravenous antibiotics are the mainstay of treatment so as to avoid laparotomy. We report on the management of two cases of postpartum OVT.

Microsatellite instability and mutation analysis of candidate genes in unselected sardinian patients with endometrial carcinoma

Microsatellite instability (MSI) is due mostly to a defective DNA mismatch repair (MMR). Inactivation of the two principal MMR genes, hMLH1 and hMSH2, and the PTEN tumor suppressor gene seems to be involved in endometrial tumorigenesis. In this study, Sardinian patients with endometrial carcinoma (EC) were analyzed to assess the prevalence of both the mutator phenotype (as defined by the presence of MSI and abnormal MMR gene expression at the somatic level) and the hMLH1, hMSH2, and PTEN germline mutations among patients with MSI positive EC.

A low-dose stimulation protocol using highly purified follicle-stimulating hormone can lead to high pregnancy rates in in vitro fertilization patients with polycystic ovaries who are at risk of a high ovarian response to gonadotropins

OBJECTIVE To study the benefits of a low-dose stimulation (LDS) protocol with purified urinary follicle-stimulating hormone in patients with polycystic ovaries who have presented previously with a very high ovarian response to a standard hMG stimulation. DESIGN Cohort study. SETTING Fertility center in a university hospital. PATIENT(S) Sixty-one patients involved in an IVF/ICSI program from January 1995 to December 1996. INTERVENTION(S) The patients were first stimulated with a standard protocol using hMG and presented with a very high ovarian response. These patients were then stimulated a second time using a low-dose protocol. Cryopreserved embryos were transferred in later artificial or natural cycles until to December 1999. MAIN OUTCOME MEASURE(S) Number of gonadotropin ampules; estradiol level on the day of ovulation induction; follicles, oocytes, and cryopreserved zygotes; fertilization, implantation, and pregnancy rates; and number of ovarian hyperstimulation syndromes (OHSS). RESULT(S) The number of ampules used, the estradiol level reached, and the number of oocytes obtained were significantly lower under the LDS than the standard protocol. High implantation (21.8%) and clinical pregnancy (38.4%) rates were obtained after LDS. The cumulated deliveries per cycle started and per patient were, respectively, 41.6% and 52.5%. Five patients suffered OHSS with the standard protocol, and none with the LDS. CONCLUSION(S) The LDS protocol offers a safe and efficient treatment for patients who present with echographic polycystic ovaries and are at risk of an excessive ovarian response to standard IVF stimulation protocols.