Charles C. Coddington

The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential * † ‡

The authors present their initial results with the hemizona assay (HZA), which was developed to predict the fertilizing potential of spermatozoa. The HZA uses the matching halves of a human zona pellucida from a nonfertilizable and nonliving oocyte, providing an internal control on zona-to-zona variability. Maximal binding of human sperm to the hemizona usually occurred after 4 to 5hours of coincubation. Sperm from fertile men exhibited significantly higher binding capacity to hemizonae compared with sperm from men who had fertilization failure during in vitro fertilization (IVF) treatment. The HZA index is calculated as follows: (bound sperm from subfertile male) ÷ (bound sperm from fertile male)×100. These findings demonstrate that the HZA may be a useful diagnostic tool in male infertility evaluations.

Factors affecting fertilization: endometrial placental protein 14 reduces the capacity of human spermatozoa to bind to the human zona pellucida*†

OBJECTIVE To examine whether placental protein 14 (PP14) may affect directly those sperm functions crucial to fertilization and early embryo development. DESIGN In these prospective studies, we evaluated semen samples of fertile men incubated under capacitating conditions with and without PP14. SETTING Academic tertiary institution. INTERVENTIONS Biologically active PP14 was purified from human midtrimester amniotic fluid by anion exchange and immunoaffinity chromatography. After separation of the motile fraction, spermatozoa were incubated for 30 minutes with or without PP14 (concentration range of 0.01 to 100 micrograms/mL), washed, and then aliquots were prepared for use in the different assays. Human sperm-zona pellucida (ZP) binding was assessed using the hemizona assay (HZA) in a 4-hour gametes coincubation period. Sperm motility parameters were evaluated using a computerized semen analyzer. The acrosome reaction (AR) was determined by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and indirect immunofluorescence. MAIN OUTCOME MEASURES Sperm-ZP binding, sperm motility patterns, and AR. RESULTS Preincubation of sperm (and not the hemizonae) with PP14 produced a significant and dose-dependent inhibition of binding in the HZA. Monoclonal antibodies generated against PP14 showed no direct effect in the HZA and partially neutralized the inhibitory activity of PP14 in the HZA. Insulin-like growth factor binding protein-1 (IGFBP-1), an endometrial stromal cell product, showed no effect in the HZA. Neither PP14 nor IGFBP-1 interfered with sperm motility parameters or the AR. CONCLUSIONS Placental protein 14 produced a potent, fast, and dose-dependent inhibition of binding of human spermatozoa to the human ZP without affecting other prefertilization events (i.e., hyperactivated motility or AR). The detrimental effect on sperm-zona interaction seems to be specific for this endometrial epithelial protein (not observed with an endometrial stromal product) and may have fundamental bearance to the fertilization process thus providing a mechanism for endometriosis-related infertility.

Evaluation of outpatient hysteroscopy, saline infusion hysterosonography, and hysterosalpingography in infertile women: a prospective, randomized study ☆

OBJECTIVE To compare the diagnostic accuracy, pain scores, and procedure length of outpatient hysteroscopy (OHS), hysterosalpingography (HSG), and saline infusion hysterosonography (SIS) for evaluation of the uterine cavity of infertile women. DESIGN Prospective, randomized, investigator-blind study. SETTING Tertiary infertility clinic. PATIENT(S) Forty-six consecutive infertile women. INTERVENTION(S) Outpatient HSG, OHS, and SIS, followed by operative hysteroscopy (HS). MAIN OUTCOME MEASURE(S) Uterine abnormalities, procedure length, and subjective pain. RESULT(S) Fifty-nine percent of infertile subjects were found to have an abnormality on at least one of three outpatient uterine evaluations. When compared with the case of definitive operative HS, 60% of abnormalities were correctly classified by HSG, 72% by OHS, and 52% by SIS (P: NS). When comparing all combinations of 2 outpatient screening tests to operative hysteroscopy, 68% were correctly classified by HSG/OHS, 58% by HSG/SIS, and 64% by OHS/SIS (P: NS). The average time length for the OHS was 9.1 min., which was significantly greater than for both HSG (average, 5.3 min) and SIS (average, 6.1 min.) (P<.0001 for both). HSG and SIS were not statistically different regarding procedure time length. The average pain score (0-10) for SIS was 2.7, compared with 5.8 and 5.3 for HSG and OHS, respectively. Both HSG and OHS mean pain scores were significantly greater than the SIS mean. CONCLUSION(S) OHS, SIS, and HSG were statistically equivalent regarding evaluation of uterine cavity pathology in infertile women.

Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome

The hemizona assay (HZA) was used in a prospective, blinded study to assess the relationship between tight sperm binding in the HZA and sperm fertilizing ability in in vitro fertilization (IVF). In each controlled assay, the authors compared sperm binding of proven fertile men with that of patients undergoing IVF. Human oocytes stored in a salt solution were used in the study, and binding results were correlated with the fertilization rate of preovulatory oocytes during IVF. Patients with poor fertilization rates in IVF had significantly lower binding than those cases with successful fertilization (7.3 +/- 1.4 versus 62.1 +/- 10.9, respectively; mean +/- standard error, P less than 0.02). Based on current standards, the HZA was able to predict fertilization accurately in 26 of 28 cases (sensitivity of 83%, specificity of 95%, positive predictive value of 83%). The authors conclude that the HZA is a valuable tool for evaluating dysfunctional sperm-zona pellucida binding, with good predictive value for fertilization in vitro.

Prediction of fertilization in vitro with human gametes : is there a litmus test ?

OBJECTIVES The objectives of this study were as follows: (1) to evaluate the relationships between sperm concentration, morphologic pattern, motion parameters, and sperm-zona pellucida binding capacity and (2) to assess their ability to predict fertilization outcome under in vitro fertilization conditions. STUDY DESIGN Semen samples from 44 infertile men were prospectively evaluated for density, morphologic pattern (strict criteria), computerized motion parameters (motility, velocity, and linearity), and hemizona assay (outcome expressed as hemizona assay index), and results were correlated with fertilization outcomes of preovulatory oocytes during in vitro fertilization. RESULTS Of all sperm parameters, morphologic pattern was the best predictor of the ability of the sperm to bind to the zona pellucida. Hemizona assay index was the best predictor of fertilization rate. Stepwise regression analysis provided a model of hemizona assay index plus motility with highest predictability (R2 = 53.4%). CONCLUSIONS (1) The influence of sperm morphologic pattern as a prognosticator of fertilization outcome is established in major part as a determinant of sperm binding ability to the zona pellucida; (2) the hemizona assay provides a robust index that is highly predictive of the potential of human gametes to achieve fertilization and highlights its use in infertility and contraception testing.

Ectopic growth of endometrium depends on its structural integrity and proteolytic activity in the cynomolgus monkey (Macaca fascicularis) model of endometriosis

OBJECTIVE To identify factors influencing the development of endometrial autografts in a monkey model of endometriosis. DESIGN Prospective, comparative study. SETTING Animal research unit. SUBJECTS Thirty regularly cycling cynomolgus monkeys in three groups of 10 each. INTERVENTIONS Endometrium was minced and spilled into the cul-de-sac in group 1. In group 2, the tissue additionally was digested enzymatically. In group 3, the tissue was incubated with a protease inhibitor. MAIN OUTCOME MEASURES Staging laparotomies after 3 weeks and 3 months. RESULTS In groups 1, 2, and 3, moderate or severe disease was seen in eight, two, and four monkeys, respectively, after 3 weeks and in eight, three, and two monkeys, respectively, at 3 months. CONCLUSIONS An intact structure leads to ectopic implantation of endometrial fragments in most cases. Conversely, enzymatic digestion of endometrial fragments and treatment with proteinase inhibitor impair ectopic growth. Intrinsic endometrial factors that influence extracellular matrix remodeling may play a role in the pathogenesis of human endometriosis.

Microlaparoscopy: a comparative study of diagnostic accuracy

OBJECTIVE To investigate the diagnostic accuracy of microlaparoscopy in comparison to laparoscopy with a standard 10-mm laparoscope. DESIGN Prospective evaluation by two independent observers. SETTING Academic Medical Center. PATIENT(S) Ten patients scheduled to undergo diagnostic laparoscopy for the indications of infertility and/or chronic pelvic pain. INTERVENTION(S) Two surgeons were present for each operation. Diagnostic laparoscopy was performed using the Microlap 2-mm laparoscope (Imagyn Medical Inc., Lagun Niguel, CA). Standard diagnostic laparoscopy was then performed using a 10-mm laparoscope. After each procedure, each surgeon reported his or her observations in a confidential manner to a third person to record. MAIN OUTCOME MEASURE(S) Endometriosis and adnexal adhesions were staged. Observations made with the microlaparoscope were compared with those obtained with a standard 10-mm laparoscope for each surgeon. The observations of both surgeons were also compared with each others to evaluate interobserver differences. RESULT(S) Operative findings reported by each individual surgeon using the microlaparoscope correlated with the operative findings reported using the larger laparoscope. Scores for both endometriosis and adnexal adhesions did not differ in any significant way. Endometriosis scores differed by no more than 6 points, and adhesion scores differed by no more than 2 points, with no subsequent change in severity classification for either finding. Furthermore, when comparing the additional operative findings of the two surgeons, no difference was noted when using either the microlaparoscope or a standard 10-mm laparoscope. CONCLUSION(S) The diagnostic accuracy achieved with microlaparoscopy is comparable to that achieved with standard 10-mm laparoscopy.

Role of hypoestrogenism or sex steroid antagonism in adhesion formation after myometrial surgery in primates

OBJECTIVE To determine the contribution of estrogen in the development of pelvic adhesions during myometrial surgery. DESIGN A randomized, prospective study in the nonhuman primate. SETTING A primate colony, Department of Obstetrics and Gynecology, Eastern Virginia Medical School. INTERVENTIONS All primates were assigned prospectively to one of three treatment groups: [1] GnRH analogue (GnRH-a), [2] mifepristone, or [3] vehicle control. After 3 months of treatment, a standard uterine fundal hysterotomy, for full thickness endometrial biopsy, was performed at the time of exploratory laparotomy, with subsequent scoring of utero-omental adhesions to the hysterotomy site at a future staging procedure based upon adhesion area, vascularity, and tenacity. Serum was drawn on the day of surgery for E2 determination. Endometrial height, from the surface interface between the endometrium and myometrium, was used as a bioassay of estrogen activity. RESULTS The hypoestrogenic (GnRH-a) group and the mifepristone group had significantly fewer utero-omental adhesions compared with the normally cycling control monkeys as measured by a lower adhesion score. Similarly, the endometrial thickness was significantly reduced in the GnRH-a and mifepristone groups (one-third) compared with the cycling controls, demonstrating the effects of either hypoestrogenism or noncompetitive estrogen antagonism. Serum E2 on the day of surgery was predictive of the postoperative adhesion score by both a regression analysis and analysis of covariance. CONCLUSIONS The actions of E2 seem to have a dramatic effect on the formation of pelvic adhesions after myometrial surgery.

Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human hemizonae pellucida after storage in a buffered salt solution

Summary. The hemizona assay (HZA) was developed to evaluate sperm binding potential using microbisected human zona pellucida. In this study, eight human oocytes stored in a buffered salt solution for 60 days were bisected into two identical hemispheres (hemizonae) and coincubated with the spermatozoa from a fertile man. All evaluated spermatozoa were tightly bound to the outer surface or had begun penetration into the zona pellucida. The hemizonae with bound spermatozoa were prepared and fixed for transmission electron microscopy (TEM) using standard techniques. Among the 108 sperm bound to the zone we were able to evaluate 25 by TEM. Twenty (80%) of the zona bound spermatozoa were partially or completely acrosome reacted, while six (20%) of the zona bound sperm had intact acrosomes. Acrosome intact, partially acrosome reacted and completely reacted spermatozoa were observed within the zona. Penetration pathways or tunnels were seen within the zona matrix. The results illustrate, that typically spermatozoa tightly bound the human zona pellucida show induction of the acrosome reaction. Importantly, following storage of human eggs in salt solution (buffered to 7.4), the zona pellucida retain their biological and functional characteristics for at least 90 days.

Defining the valid hemizona assay: accounting for binding variability within zonae pellucidae and within semen samples from fertile males

OBJECTIVE To achieve a better understanding of the variability in sperm and oocyte binding capacities will optimize use of the hemizona assay (HZA) as a predictor of sperm function. DESIGN Limitations of the HZA were more clearly delineated by current studies: (1) variability of sperm binding capacity of men over a 90-day interval; (2) variability of sperm binding using different oocytes; and (3) lower limits of the number of sperm bound from the fertile control in two laboratories. PATIENTS Semen was obtained from proven fertile men and one subfertile individual. MAIN OUTCOME MEASURE The number of sperm tightly bound to the hemizona were measured and compared. RESULTS In the initial study, 6 fertile control men exhibited a similar degree of variability in zona binding when studied over a 90-day interval. Average sperm binding for individuals ranged from 68 to 127. Second, 3 of the 15 simultaneous assays showed very low numbers of sperm bound, indicating that 20% of the zonae had poor binding. Third, from 18 men who had 0% fertilization in an in vitro fertilization system using mature oocytes, evaluation of their sperm by HZA was performed. The sperm bound poorly and the 95% confidence interval was 20 sperm bound. Thus, the fertile controls should bind greater than 20 sperm to distinguish them from the infertile group in the HZA system resulting in a valid assay. CONCLUSIONS With these guidelines, applications of the HZA may be made with greater reassurance of a valid bioassay of sperm fertilizing potential.