[Post-thaw sperm functions of high- and low-fertility sperm donors: a comparative study of 40 cases].

OBJECTIVE: To investigate sperm function indexes that can be used to effectively evaluate the sperm donors' fertility so as to select healthy post-thaw semen samples and improve the success rate of assisted reproductive technology. METHODS: According to the pregnancy outcomes, we divided 40 donor semen samples into a high-fertility group (n = 20) and a low-fertility group (n = 20). We measured and compared the concentration, progressive motility, morphology, acrosome intactness, DNA integrity and mitochondrial membrane potential (MMP) of the post-thaw sperm between the two groups. RESULTS: There were statistically significant differences between the high- and low-fertility groups in the percentages of morphologically normal sperm ([18.50 +/- 6.10]% vs [14.42 +/- 6.44]%, P < 0.01), acrosome intactness ([86.17 +/- 4.49]% vs [80.04 +/- 7.52]%, P < 0.05) and DNA fragmentation index ([9.21 +/- 3.22]% vs [15.72 +/- 8.20]%, P < 0.05), but not in MMP ([56.75 +/- 18.80]% vs [52.23 +/- 18.86]%, P > 0.05). A significantly positive correlation was found between MMP and sperm motility (r = 0.760, P < 0.05), but not between other sperm functions and sperm concentration and motility. CONCLUSION: Sperm concentration, motility, morphology, acrosome intactness rate and DNA integrity contribute effectively to the evaluation of the fertilization capacity of post-thaw donor semen samples.

[Pre-freezing equilibration improves post-thaw human semen parameters].

OBJECTIVE: To investigate the effect of pre-freezing equilibration on the cryo-survival of human sperm and to optimize the protocol of direct fumigation for the freeze-thawing of human sperm. METHODS: We collected 50 semen samples from healthy donors, each subjected to cryopreservation with 3 different methods: non-equilibration freezing (Group A), 10-min equilibration at room temperature before freezing (Group B), and 10-min equilibration at 4 degrees C before freezing (Group C). We examined all the post-thaw semen samples by computer-assisted semen analysis for the sperm motility parameters, and detected the sperm vitality and deformity index (SDI). RESULTS: The recovery rate of progressive sperm motility was (61.88 +/- 16.94)% in Group C, remarkably higher than in A ([48.61 +/- 16.44]%) and B ([49.41 +/- 13.77]%) (P < 0.05), but with no significant difference between the latter two. And there were no significant differences in sperm vitality and SDI among the three groups. CONCLUSION: Ten-minute equilibration at 4 degrees C before freezing can evidently improve the progressive motility of sperm in addition to its advantages of easy operation and controllable experimental condition.

[Three-step sperm retrieval for 73 non-obstructive azoospermia patients].

OBJECTIVE: To investigate the value and clinical application of the Three-Step Sperm Retrieval method in improving the sperm retrieval rate for non-obstructive azoospermia (NOA) patients. METHODS: Seventy-three NOA patients underwent Three-Step Sperm Retrieval in the following order of procedures: testicular fine needle aspiration (TFNA), testicular sperm extraction (TSE), and microdissection testicular sperm extraction (MD-TSE). The testicular tissue obtained from each step was observed for spermatozoa under the 400-fold inverted microscope. If spermatozoa were found in one step, the operation would be stopped; otherwise, the next step would be carried out. The testicular tissue was subjected to pathological examination. RESULTS: Spermatozoa was found in the testicular tissue in 38.4% of the cases (28/73) at TFNA as the first step, in 52.1% (38/73) at TFNA and TSE, and in 64.4% (47/73) at TFNA, TSE and MD-TSE. Pathological examination showed 25 of the cases to be Sertoli cell-only syndrome, 21 to be sperm maturation arrest and the other 27 to be hypospermatogenesis, in which spermatozoa were found in 10, 14 and 23 cases, respectively. CONCLUSION: The Three-Step Sperm Retrieval method can significantly improve the sperm retrieval rate for NOA patients. And the sperm retrieval rate is associated with the pathological type of the testicular tissue, a higher rate with hypospermatogenesis.

[Reference values of semen parameters for normal fertile men in Shanghai].

OBJECTIVE: To analyze the distribution characteristics of the main semen parameters of healthy semen donors and normal fertile men in Shanghai, compare the semen quality between the two groups, and investigate the normal reference values of the semen parameters of the fertile population in Shanghai. METHODS: We obtained semen samples from 100 healthy donors and 41 fertile men, performed semen analyses according to the WHO (2010) guidelines, and determined the semen volume, sperm concentration, sperm progressive motility, total sperm count and total progressively motile sperm count. We analyzed the distribution of the semen parameters of the normal fertile men, and obtained the lower limits of their normal reference values. RESULTS: There were no statistically significant differences in the main semen parameters between the healthy donors and normal fertile men (P < 0.05). The lower reference limits for the semen parameters of normal fertile men in Shanghai (P < 0.05) were as follows: sperm concentration > or = 27.3 x 10(6)/ml, sperm progressive motility > or = 8.1%, semen volume > or = 0.82 ml, total sperm count > or = 44.73 x 10(6) per ejaculate, and total progressively motile sperm count > or = 24.68 x 10(6) per ejaculate. CONCLUSION: For the evaluation of male fecundity, total sperm count and total progressively motile sperm count may be two better predictors than others.

[Desert hedgehog regulates the proliferation and differentiation of Leydig cells: an update].

Hedgehog signaling pathway plays an important role in regulating the development of endocrine glands. Desert hedgehog (Dhh) has become a recent focus for its regulation of testis development, especially of Leydig cells. Dhh, as a Sertoli cell product, regulates the proliferation and differentiation of Leydig cell lineage and functions to secrete testosterone through a paracrine signaling mechanism. Testosterone, as the most important sex hormone of the male, plays a critical role in testis development, spermatogenesis and maintenance of normal masculinization. Therefore, normal Dhh signaling pathway ensures normal spermatogenic function. Researches on the Hedgehog signaling pathway in the testis have a potential significance for studying the pathophysiological mechanisms of androgen deficiency and dyszoospermia, as well as for the clinical treatment of these diseases.

[Direct fumigation for freeze-thawing of human sperm: an experimental study].

OBJECTIVE: To study the effect of direct fumigation on the post-thaw recovery rate of cryopreserved spermatozoa, and to search for a best method for human sperm cryopreservation. METHODS: We collected semen samples from 100 donors conforming to the normal reference values in WHO Laboratory Manual for the Examination and Processing of Human Semen (5th Ed), divided them into two groups, and subjected them to cryopreservation by programmable freezing (Group A) and direct fumigation (Group B), respectively. We detected the progressive motility of pre-freezing and post-thaw sperm with a computer-assisted semen analyzer, and compared the effects of the two methods on the functional integrity of sperm membrane and the rate of abnormal sperm using the percentage of hypo-osmotic swelling sperm and modified Papanicolaou staining. RESULTS: Statistically significant differences were found in post-thaw sperm progressive motility between the Groups A and B ([34.0 +/- 18.4]% vs [43.0 +/- 19.5]%, P<0.05), both remarkably decreased as compared with pre-freezing ([57.0 +/- 16.7]%, P<0.05). Such differences were also found in the post-thaw recovery rate of progressively motile sperm between the two groups ([52.2 +/- 20.6]% vs [67.1 +/- 20.0]%, P<0.05). The post-thaw percentage of hypo-osmotic swelling sperm was obviously decreased in both Groups A and B ([67.1 +/- 11.1]% and [70.6 +/- 10.0]%) in comparison with pre-freezing ([84.5 +/- 7.5]%, P<0.05), with significant differences between A and B (P<0.05). However, the rate of sperm abnormality was evidently increased in Groups A and B ([85.0 +/- 8.7% and [85.7 +/- 9.1]%), significantly higher than pre-freezing ([77.8 +/- 9.6]%, P<0.05), but with no significant differences between A and B (P>0.05). CONCLUSION: Direct fumigation is superior to programmable freezing for its easier operation, wider application, and higher sperm recovery rate.

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study.

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.

[Reconstructed seminiferous tubules from ectopic grafting of testicular cells].

OBJECTIVE: To investigate the ectopic grafts of mouse testicular cells by observing the reconstruction of seminiferous tubules, colonization of spermatogenic cells and spermatogenesis using immunodeficient mice as recipients. METHODS: The testes of newborn male ICR mice were digested to obtain single cell suspension. The cells were then mixed with matrigel and subcutaneously grafted into the dorsal region of the male nude mice. The mice were castrated after the operation and the grafts were dissected from 5 of the nude mice at 4, 6, 8 and 10 weeks, respectively. The success rates of transplantation and the graft diameters were calculated, and the structure of the reconstituted seminiferous tubules, colonization of the germ cells and spermatogenesis were observed by HE staining and immunohistochemistry. RESULTS: All the mice recipients survived after the testicular cell transplantation. Within 10 weeks after the operation, tissue masses could be observed, with the diameter increased from (3.91 +/- 0.71) mm at 4 weeks to (6.69 +/- 0.50) mm. Neovascularization was detected at the surface of the masses and seminiferous tubule structures found in the grafts. The germ cells that developed from spermatogonia to round spermatids were observed, but with no sperm in the tubules. Germ cells, Sertoli cells and Leydig cells were identified by immunochemical detection of Mvh, Gata4 and P450Scc in the grafts at 8 weeks. CONCLUSION: Seminiferous tubules could be ectopically reconstructed from suspension of neonatal mouse testicular cells. Ectopic grafting provided a preferable model for the studies on testis tissue engineering and interactions between testicular cells during testicular development and spermatogenesis.

[Advances in the study of the differentiation of stem cells into male gametes].

male gametes play a critical role in transmitting hereditary information to the offspring. Gametogenesis, especially spermatogenesis, is a complicated differentiation process including mitosis and meiosis. However, for lack of an efficient and reproducible model, the mechanism of male germ development is not yet clear. Researches on stem cells'differentiation into male gametes in vitro will promote the study of germ cell development and even reproductive biology. This article updates the researches on the culture of primordial germ cells and stem cells' differentiation into male gametes.

[Safety evaluation of the offspring conceived by assisted reproductive technology with donor's semen].

OBJECTIVE: To investigate and compare the incidences of birth defects in the offspring conceived by assisted reproductive technology (ART), including artificial insemination with the donor's semen (AID), in vitro fertilization with donor's semen (IVF-D) and intracytoplasmic sperm injection with the donor's semen (ICSI-D), and in those conceived through ART with the husband's semen, including AIH, IVF and ICSI, in order to further evaluate the safety of ART with the donor's semen. METHODS: From January 2005 to October 2009, Shanghai Human Sperm Bank provided sperm copies to 11 medical institutions, which resulted in a total of 904 offspring born by ART. We followed up all these cases and investigated the status of the offspring. The control group included 4195 offspring of infertile couples from 4 Reproductive Medical Centers approved by health management administrations, which were conceived by ART in the same period. After investigating the number of offspring and cases of birth defects caused by various methods of assisted reproductive technology, we compared the incidence of birth defects resulting from the donor's semen and that from the husband's. RESULTS: There were 7 cases of birth defects (0.77%) in the offspring born by ART with the donor's semen, and 42 cases (1.00%) in those born by ART with the husband's semen, with no significant differences between the two groups (P > 0.05). CONCLUSION: There were no significant differences in the category of birth defects between ART with the donor's semen and that with the husband's, while the incidence of birth defects resulting from ART with the donor's semen was significantly lower than that from ICSI in infertile couples. The present findings indicate a higher safety of ART with the donor's semen.

[Spontaneous differentiation potency of induced pluripotent stem cells into male germ cells in vitro].

OBJECTIVE: To explore the expression profile of male germ cell-associated genes during the spontaneous differentiation of induced pluripotent stem cells (iPS) and assess the potency of their spontaneous differentiation into male germ cells in vitro. METHODS: Embryoid body (EB) formation was used to promote the spontaneous differentiation of iPS into male germ cells, and the expressions of germ cell-associated genes were detected by real-time PCR and PCR. RESULTS: Real-time PCR and PCR revealed different expression levels of relevant genes at different times of iPS spontaneous differentiation into male germ cells. Each of the 9 genes analyzed exhibited one of the four temporal expression patterns: wavelike increase of Oct4, progressive decrease of Dppa3 and Stra8, wavelike decrease of Dazl, and decrease following initial increase of Tex14, Msy2, Scp1, Scp3 and Akap3. CONCLUSION: Induced pluripotent stem cells express male germ cell-associated genes and male haploid genes during their spontaneous differentiation through EB formation, and have the potency of differentiating into male gametes.

[Construction of recombinant pEGFP/hTRT plasmid].

OBJECTIVE: To construct a plasmid which has a reporter gene for exploring the role of human telomerase reverse transcriptase(hTRT) in in-vitro cell cultivation. METHODS: hTRT was cut by restricted enzyme from plasmid pGRN145 and inserted to plasmid pEGFP-C1 (enhanced green fluorescent protein). RESULTS: Restricted enzyme analysis and DNA sequencing showed that the sequence of the pEGFP -hTRT transgenic plasmid was correct. CONCLUSION: The recombinant vector pEGFP-hTRT has been successfully constructed, and it can be used as a transgenic plasmid in generating immortalized cell lines.

[The cloning of mouse FasL gene and expression in chondrocytes].

AIM: To clone mFasL cDNA, then insert it into retrovirus expression system pLNCX(2), and express mFasL cDNA in chondrocytes to study its function. METHODS: RT-PCR was applied to amplify mFasL cDNA from the total RNA of activated mouse spleen lymphocytes. The cDNA was inserted into a T-cloning vector, then subcloned into pLNCX(2). After transfection of chondrocytes, expression of FasL cDNA was detected by FACS, and activity of expressed product was analyzed by single mixed lymphocyte reaction(SMLC). RESULTS: mFasL cDNA fragment of 0.855 kb was successfully cloned and verified by sequencing. The transfection efficiency of mFasL cDNA in chondrocytes determined by FACS was 60.64%. Expressed product could evidently inhibit SMLC of allogeneic lymphocytes. It's stimulating index (SI) reduced to 11.71% as compare with the untransfected chondrocytes. CONCLUSION: Cloned recombinant pLNCX(2)-FasL can effectively express mFasL in chondrocytes, and can evidently inhibit SMLC of allogeneic lymphocytes.