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Initiators of leiomyoma: differential proto-oncogene expression in small versus large fibroids identified by microarray analysis
Materials and Methods: Immunohistochemistry was performed on paraffin sections of archived tissues and SKOV3 ovarian cancer cell lines. Effects of antibodies to RCP on SKOV3 ovarian cancer cells in tissue culture were investigated. Results: RCP was localized to the cytoplasm of uterine endometrial glands. In the normal uterus and endometrial hyperplacia RCP was present in the cells of the glandular epithelium and presented polarity, being localized to the apical part of the cells. In advanced uterine adenocarcinoma, this polarity was lost. In the term placenta, RCP was localized to the villi as well as the endothelium of the blood vessels. RCP also could be localized to the cytoplasm of ovarian epithelial adenocarcinoma cells and cultured SKOV3 cells. Antibodies against RCP inhibited uptake of tritiated riboflavin by these cells. Addition of polyclonal antibodies against RCP to SKOV3 cells in culture caused loss of attachment and cell death by 24 to 48 hrs. Preliminary results have revealed localization of RCP to the cell membrane of SKOV3 cells. Conclusion: Both the ovary and the uterus are prime targets for estrogen action and these studies indicate a possible essential role for RCP in mediating estrogen action. Supported by: a grant from NCI, 5RO3CA91185-2.
P-389 How often does transperitoneal transmigration of sperm occur in effecting human pregnancy? Gerard G. Nahum, Connette McMahon, Harold Stanislaw. Duke Univ Sch of Medicine, Durham, NC; CA State Univ, Stanislaus, Turlock, CA. Objective: To determine how often sperm transmigrates across the peritoneal cavity to effect intrauterine human pregnancy. Design: Two-hundred seventy-two women were identified with noncommunicating rudimentary uterine horn pregnancies who had prior parity data available. All pregnancies in the “blind” rudimentary horns had no direct communication to the cervix or vagina and necessarily resulted from transperitoneal transmigration of either sperm or fertilized ova. All prior clinical pregnancies occurred in the unicornuate hemi-uterus with communication to the cervix. In 187 cases, the population fertility rate that was specific to each patient’s age, era, and country of origin was also available, such that the expected prior parity for each woman could be calculated. Materials and Methods: Between 1900-1999, 272 women with noncommunicating uterine horn pregnancies were identified in the world literature who had prior pregnancy information. The total number of observed pregnancies prior to the incident rudimentary horn pregnancies was determined. For a subset of 187 women who had age, country, and era-specific fertility rate information available, the total number of expected prior pregnancies was also calculated. The proportional rudimentary horn pregnancy results were used as upper and lower bounds to infer the per pregnancy sperm transperitoneal transmigration rate. The actual prior parity frequency distribution for the 272 women was also determined and the best-fit per pregnancy probability of attaining a rudimentary horn pregnancy was calculated using the method of maximum likelihood estimation. Results: The total number of observed pregnancies prior to the incident 272 rudimentary horn pregnancies was 292, yielding a ratio of actual rudimentary to non-rudimentary uterine horn pregnancies of 0.93. For the subset of 187 women who had age, country, and era-specific fertility rate information available, the total number of actual prior pregnancies was 166 and the total number of expected prior pregnancies was 218 (ratios of rudimentary to non-rudimentary horn pregnancies of 1.13 and 0.86, respectively). These proportional rudimentary horn pregnancy results directly imply maximum and minimum per pregnancy sperm transmigration rates of 0.53 and 0.46, respectively. The actual prior parity frequency distribution for all 272 women was P0⫽136, P1⫽67, P2⫽30, P3⫽18, P4⫽8, P5⫽3, P6⫽8, P7⫽1, P8⫽0, and P9⫽1. Using maximum likelihood estimation, the best-fit probability of attaining a rudimentary horn gestation per pregnancy was 0.51 (Figure), with a range from 0.48 to 0.56 (p ⬎0.05 in all cases, indicating no statistically significant difference between the actual prior parity distribution and the calculated parity distribution over this range).
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Abstracts
Conclusion: Intraperitoneal sperm transmigration occurs approximately half the time in effecting human pregnancy. In view of this result, it can be suggested that in order to minimize the risk of ectopic tubal pregnancy in cases where a woman has a unilaterally damaged fallopian tube and a normal contralateral salpinx, salpingectomy is the preferred surgical treatment rather than attempting tubal salvage and repair. P-390 Initiators of leiomyoma: Differential proto-oncogene expression in small versus large fibroids identified by microarray analysis. William H. Catherino, Phyllis C. Leppert, Mark Payson, Clariss Potlog-Nahari, Lynnette K. Nieman, James H. Segars. National Inst of Health, Bethesda, MD. Objective: The fundamental genetic alterations contributing to early fibroid development have not been characterized. Identification of such genes may provide clues to fibroid etiology and may provide insight into possible treatments. In order to investigate early steps in fibroid development, very small fibroids must be studied. Without clear direction as to which genes to investigate, the limited amounts of tissue have inhibited a survey of candidate genes. We performed microarray analysis with confirmatory reverse transcriptase-polymerase chain reaction (RT-PCR) to identify genes that may be important for early fibroid development. Design: Screening microarray analysis followed by confirmatory RTPCR. Materials and Methods: After IRB approval, we collected specific fibroids from patients who were undergoing abdominal hysterectomy. In this study, six fibroids were collected that ranged in size from 0.2cm-0.5cm. The comparison fibroid from the same patient was 2.5 by 1.2 by 2.1 cm. RNA isolation was performed using a standard protocol. Samples of 10-15 micrograms of total RNA from fibroid and adjacent myometrium were analyzed by Affymetrix U133 microarray chips. For RT-PCR, primers were designed and added to the reaction mix at 200nM concentration. RT-PCR with Platinum Taq was performed, and primers for GAPDH were included as an internal control. Samples were run on a 2% agarose gel with ethydium bromide. RT-PCR confirmation was also performed on two fibroids from the primary patient as well as from fibroids of four non-related patients. Results: Microarray Data are presented in table form below. These proto-oncogenes were also over-expressed in small fibroids compared to surrounding myometrium. RT-PCR Demonstrated over-expression of above proto-oncogenes in small fibroids compared to large fibroids from this and other patients.
Vol. 80, Suppl. 3, September 2003
Conclusion: Our findings indicate that proto-oncogenes involved in early response to inflammation and cell stress were over-expressed early fibroids. Over-expression of such genes in early fibroids provides a link between hormone function (particularly estrogen action) and dysregulated gene expression. These proto-oncogenes also link early fibroid development with alterations seen in the initiation of neoplasia.
P-391 Sperm deformity index: Correlation with quality and apoptotic markers of ejaculated spermatozoa. Nabil Aziz, Tamer M. Said, Rakesh K. Sharma, Sreedhar Rama, Anthony J. Thomas Jr., Ashok Agarwal. Liverpool Women’s Hosp, Liverpool, United Kingdom; Cleveland Clin Fdn, Cleveland, OH. Objective: Apoptosis has been implicated as a cause of male infertility. Spermatozoa exhibiting apoptotic signals are often present in the ejaculate. Semen analysis including the assessment of sperm morphology and the sperm deformity index (SDI) remain standard procedures for evaluating the fertility potential. The objective of our study was to assess the correlation of SDI with parameters of sperm quality and apoptotic markers of ejaculated spermatozoa. Design: Prospective-controlled study. Materials and Methods: Semen specimens were collected from 22 infertile men and from 5 healthy donors with normal semen parameters who served as controls. Sperm count and motility was assessed according to the World Health Organization guidelines. Smears of fresh semen were prepared for assessment of sperm morphology applying the strict criteria. SDI was calculated by dividing the total number of deformities observed by the number of sperm randomly selected and evaluated, irrespective of their morphological normality. Annexin V/propidium iodide staining was carried out in order to measure the percentage of apoptotic and necrotic sperm. Two hundred spermatozoa were randomly assessed per slide in five fields and scored as intact, apoptotic or necrotic. Results: Median and interquartile values (25thand 75th percentiles) of semen parameters, acrosomal damage and SDI scores in donors and infertile men are shown in the table. Donors’ had significantly higher proportions of count, motility and morphologically normal sperm; in addition to significantly lower proportions of sperm with damaged acrosomes, SDI scores and percentage of apoptotic and necrotic sperm, compared to infertile men. The proportion of apoptotic ⫹ necrotic sperm positively correlated with SDI (r ⫽ 0.46, P ⫽ 0.04), however, it did not correlate with the percent apoptotic sperm per se. When donors ⫹ patients were combined, an overall significant inverse correlation was observed between normal SDI and sperm concentration (r ⫽ -0.72; P ⬍ 0.04); motility (r ⫽ -0.62; P ⬍ 0.002); and normal morphology (r ⫽ -0.54; P ⬍ 0.01). A higher percentage of acrosomal damage was seen in samples with high proportions of necrotic sperm (r ⫽ 0.45, P ⫽ 0.03).
Nieman, James H. Segars. National Inst of Health, Bethesda, MD; Univ of South Florids, Tampa, FL. Objective: Uterine leiomyoma place a significant burden on those afflicted as well as on the United States health care system. Fibroids are made up of disordered collagenous tissue and are more prevalent in African Americans compared to Caucasian Americans. Keloids are similarly made up of disordered collagenous tissue and are also more prevalent in African Americans. Despite these similarities, there is no known molecular link between these two diseases. Dermatopontin is a 22KDa extra-cellular protein that can interact with two cell signaling pathways: the integrin system and the TGF  system. Dermatopontin expression is decreased in hypertrophic scars (Kuroda et al., 1999) and in fibroids compared to myometrium using microarray analysis (Tsibris et al., 2002). It is therefore possible that differential expression of dermatopontin is a link between the fibroid and keloid phenotype. Design: Screening microarray analysis of 33,000 genes followed by confirmatory reverse transcriptase PCR, real time RT-PCR, light and electron microscopy. Materials and Methods: After IRB approval, we evaluated the expression of dermatopontin in multiple fibroids and associated myometrium in twenty four samples from a total of eleven patients. Microarray studies using Affymetrix U133 chips were used to identify differential gene expression between fibroid and myometrium. Differentially-expressed genes were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), and real-time RT-PCR was used to quantitate differential expression. Light and electron microscopy evaluation were used to demonstrate differential collagen formation between fibroid and myometrium. Results: Dermatopontin mRNA expression was decreased in fibroids compared to myometrium in our microarray experiments (see Table 1). This finding was confirmed by semi-quantitative RT-PCR and real time RT-PCR (dermatopontin under-expression of 0.11 (95% CI⫽[0.09,0.13])). Associated genes such as decorin, IL-4 and TGF 1 were not differentially expressed by semi-quantitative RT-PCR, while TGF 3 was over-expressed in fibroids compared to control myometrium. Light and electron microscopy demonstrated disordered collagen formation similar to keloid tissue.
Conclusion: Our results suggest that under-expression of dermatopontin may play an important role in both fibroid and keloid formation.Decreased dermatopontin may alter both integrin and TGF 3 signaling. Dermatopontin may thereby provide a promoting stimulus for leiomyoma growth. P-393 The correlation of leptin and hCG (Human Chorionic Gonadotrophin) levels in the serum between women with hyperemesis gravidarum and normal control. Jiae Lee, Kyung-Hee Lee, Min-Sik Kim, Bum-Chae Choi, Keun-Jae Yoo, Jeong-Wook Kim. Lab of Reproductive Medicine, Creation & Love Women’s Hosp, Gwangju, Republic of Korea; Ctr for Recurrent Miscarriage and Infertility, Creation & Love Women’s Hosp, Gwangju, Republic of Korea; Dept. of Ob/Gyn, Samsung Cheil Hosp, Sungkyunkwan Univ, Sch of Medicine, Seoul, Republic of Korea.
P ⬍0.05 significance by Mann-Whitney U test. Conclusions: Poor sperm morphology and high SDI are associated with high proportions of apoptotic ⫹ necrotic sperm in ejaculated semen. Thus, the role of morphological assessment cannot be ruled out as a predictor of apoptosis in spermatozoa.
P-392 Promoters of leiomyoma: Dermatopontin expression in leiomyoma suggests an association between fibroids and keloids. William H. Catherino, Matthew H. Stenmark, Phyllis C. Leppert, John C. M. Tsibris, Lynnette K.
FERTILITY & STERILITY威
Objective: Hyperemesis gravidarum is a distinct feature occurring in 0.2-1% of all pregnant women and defined as severe form of nausea and vomiting that lead to fluid and electrolyte imbalance, ketonuria, and weight loss during early pregnancy. Hyperemesis gravidarum may be associated with the development or worsening of an eating disorder during pregnancy. Leptin is the 16kDa protein product of the obese gene, which limits food intake and increases energy expenditure and is produced by the human placenta during pregnancy. High leptin level may decrease food intake and metabolic efficiency. The purpose of this study is to evaluate the correlation of leptin and hCG level in the serum of patients with hyperemesis gravidarum compared with a normal control group. Design: Analysis of serum leptin level by sandwich ELISA and serum hCG level by RIA. Materials and Methods: The serum were collected from 16 women with hyperemesis gravidarum and 10 women with normal pregnancy. They were at the gestation stages of 6-9 weeks. The serum leptin and hCG were
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P-389 How often does transperitoneal transmigration of sperm occur in effecting human pregnancy? Gerard G. Nahum, Connette McMahon, Harold Stanislaw. Duke Univ Sch of Medicine, Durham, NC; CA State Univ, Stanislaus, Turlock, CA. Objective: To determine how often sperm transmigrates across the peritoneal cavity to effect intrauterine human pregnancy. Design: Two-hundred seventy-two women were identified with noncommunicating rudimentary uterine horn pregnancies who had prior parity data available. All pregnancies in the “blind” rudimentary horns had no direct communication to the cervix or vagina and necessarily resulted from transperitoneal transmigration of either sperm or fertilized ova. All prior clinical pregnancies occurred in the unicornuate hemi-uterus with communication to the cervix. In 187 cases, the population fertility rate that was specific to each patient’s age, era, and country of origin was also available, such that the expected prior parity for each woman could be calculated. Materials and Methods: Between 1900-1999, 272 women with noncommunicating uterine horn pregnancies were identified in the world literature who had prior pregnancy information. The total number of observed pregnancies prior to the incident rudimentary horn pregnancies was determined. For a subset of 187 women who had age, country, and era-specific fertility rate information available, the total number of expected prior pregnancies was also calculated. The proportional rudimentary horn pregnancy results were used as upper and lower bounds to infer the per pregnancy sperm transperitoneal transmigration rate. The actual prior parity frequency distribution for the 272 women was also determined and the best-fit per pregnancy probability of attaining a rudimentary horn pregnancy was calculated using the method of maximum likelihood estimation. Results: The total number of observed pregnancies prior to the incident 272 rudimentary horn pregnancies was 292, yielding a ratio of actual rudimentary to non-rudimentary uterine horn pregnancies of 0.93. For the subset of 187 women who had age, country, and era-specific fertility rate information available, the total number of actual prior pregnancies was 166 and the total number of expected prior pregnancies was 218 (ratios of rudimentary to non-rudimentary horn pregnancies of 1.13 and 0.86, respectively). These proportional rudimentary horn pregnancy results directly imply maximum and minimum per pregnancy sperm transmigration rates of 0.53 and 0.46, respectively. The actual prior parity frequency distribution for all 272 women was P0⫽136, P1⫽67, P2⫽30, P3⫽18, P4⫽8, P5⫽3, P6⫽8, P7⫽1, P8⫽0, and P9⫽1. Using maximum likelihood estimation, the best-fit probability of attaining a rudimentary horn gestation per pregnancy was 0.51 (Figure), with a range from 0.48 to 0.56 (p ⬎0.05 in all cases, indicating no statistically significant difference between the actual prior parity distribution and the calculated parity distribution over this range).
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Abstracts
Conclusion: Intraperitoneal sperm transmigration occurs approximately half the time in effecting human pregnancy. In view of this result, it can be suggested that in order to minimize the risk of ectopic tubal pregnancy in cases where a woman has a unilaterally damaged fallopian tube and a normal contralateral salpinx, salpingectomy is the preferred surgical treatment rather than attempting tubal salvage and repair. P-390 Initiators of leiomyoma: Differential proto-oncogene expression in small versus large fibroids identified by microarray analysis. William H. Catherino, Phyllis C. Leppert, Mark Payson, Clariss Potlog-Nahari, Lynnette K. Nieman, James H. Segars. National Inst of Health, Bethesda, MD. Objective: The fundamental genetic alterations contributing to early fibroid development have not been characterized. Identification of such genes may provide clues to fibroid etiology and may provide insight into possible treatments. In order to investigate early steps in fibroid development, very small fibroids must be studied. Without clear direction as to which genes to investigate, the limited amounts of tissue have inhibited a survey of candidate genes. We performed microarray analysis with confirmatory reverse transcriptase-polymerase chain reaction (RT-PCR) to identify genes that may be important for early fibroid development. Design: Screening microarray analysis followed by confirmatory RTPCR. Materials and Methods: After IRB approval, we collected specific fibroids from patients who were undergoing abdominal hysterectomy. In this study, six fibroids were collected that ranged in size from 0.2cm-0.5cm. The comparison fibroid from the same patient was 2.5 by 1.2 by 2.1 cm. RNA isolation was performed using a standard protocol. Samples of 10-15 micrograms of total RNA from fibroid and adjacent myometrium were analyzed by Affymetrix U133 microarray chips. For RT-PCR, primers were designed and added to the reaction mix at 200nM concentration. RT-PCR with Platinum Taq was performed, and primers for GAPDH were included as an internal control. Samples were run on a 2% agarose gel with ethydium bromide. RT-PCR confirmation was also performed on two fibroids from the primary patient as well as from fibroids of four non-related patients. Results: Microarray Data are presented in table form below. These proto-oncogenes were also over-expressed in small fibroids compared to surrounding myometrium. RT-PCR Demonstrated over-expression of above proto-oncogenes in small fibroids compared to large fibroids from this and other patients.
Vol. 80, Suppl. 3, September 2003
Conclusion: Our findings indicate that proto-oncogenes involved in early response to inflammation and cell stress were over-expressed early fibroids. Over-expression of such genes in early fibroids provides a link between hormone function (particularly estrogen action) and dysregulated gene expression. These proto-oncogenes also link early fibroid development with alterations seen in the initiation of neoplasia.
P-391 Sperm deformity index: Correlation with quality and apoptotic markers of ejaculated spermatozoa. Nabil Aziz, Tamer M. Said, Rakesh K. Sharma, Sreedhar Rama, Anthony J. Thomas Jr., Ashok Agarwal. Liverpool Women’s Hosp, Liverpool, United Kingdom; Cleveland Clin Fdn, Cleveland, OH. Objective: Apoptosis has been implicated as a cause of male infertility. Spermatozoa exhibiting apoptotic signals are often present in the ejaculate. Semen analysis including the assessment of sperm morphology and the sperm deformity index (SDI) remain standard procedures for evaluating the fertility potential. The objective of our study was to assess the correlation of SDI with parameters of sperm quality and apoptotic markers of ejaculated spermatozoa. Design: Prospective-controlled study. Materials and Methods: Semen specimens were collected from 22 infertile men and from 5 healthy donors with normal semen parameters who served as controls. Sperm count and motility was assessed according to the World Health Organization guidelines. Smears of fresh semen were prepared for assessment of sperm morphology applying the strict criteria. SDI was calculated by dividing the total number of deformities observed by the number of sperm randomly selected and evaluated, irrespective of their morphological normality. Annexin V/propidium iodide staining was carried out in order to measure the percentage of apoptotic and necrotic sperm. Two hundred spermatozoa were randomly assessed per slide in five fields and scored as intact, apoptotic or necrotic. Results: Median and interquartile values (25thand 75th percentiles) of semen parameters, acrosomal damage and SDI scores in donors and infertile men are shown in the table. Donors’ had significantly higher proportions of count, motility and morphologically normal sperm; in addition to significantly lower proportions of sperm with damaged acrosomes, SDI scores and percentage of apoptotic and necrotic sperm, compared to infertile men. The proportion of apoptotic ⫹ necrotic sperm positively correlated with SDI (r ⫽ 0.46, P ⫽ 0.04), however, it did not correlate with the percent apoptotic sperm per se. When donors ⫹ patients were combined, an overall significant inverse correlation was observed between normal SDI and sperm concentration (r ⫽ -0.72; P ⬍ 0.04); motility (r ⫽ -0.62; P ⬍ 0.002); and normal morphology (r ⫽ -0.54; P ⬍ 0.01). A higher percentage of acrosomal damage was seen in samples with high proportions of necrotic sperm (r ⫽ 0.45, P ⫽ 0.03).
Nieman, James H. Segars. National Inst of Health, Bethesda, MD; Univ of South Florids, Tampa, FL. Objective: Uterine leiomyoma place a significant burden on those afflicted as well as on the United States health care system. Fibroids are made up of disordered collagenous tissue and are more prevalent in African Americans compared to Caucasian Americans. Keloids are similarly made up of disordered collagenous tissue and are also more prevalent in African Americans. Despite these similarities, there is no known molecular link between these two diseases. Dermatopontin is a 22KDa extra-cellular protein that can interact with two cell signaling pathways: the integrin system and the TGF  system. Dermatopontin expression is decreased in hypertrophic scars (Kuroda et al., 1999) and in fibroids compared to myometrium using microarray analysis (Tsibris et al., 2002). It is therefore possible that differential expression of dermatopontin is a link between the fibroid and keloid phenotype. Design: Screening microarray analysis of 33,000 genes followed by confirmatory reverse transcriptase PCR, real time RT-PCR, light and electron microscopy. Materials and Methods: After IRB approval, we evaluated the expression of dermatopontin in multiple fibroids and associated myometrium in twenty four samples from a total of eleven patients. Microarray studies using Affymetrix U133 chips were used to identify differential gene expression between fibroid and myometrium. Differentially-expressed genes were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), and real-time RT-PCR was used to quantitate differential expression. Light and electron microscopy evaluation were used to demonstrate differential collagen formation between fibroid and myometrium. Results: Dermatopontin mRNA expression was decreased in fibroids compared to myometrium in our microarray experiments (see Table 1). This finding was confirmed by semi-quantitative RT-PCR and real time RT-PCR (dermatopontin under-expression of 0.11 (95% CI⫽[0.09,0.13])). Associated genes such as decorin, IL-4 and TGF 1 were not differentially expressed by semi-quantitative RT-PCR, while TGF 3 was over-expressed in fibroids compared to control myometrium. Light and electron microscopy demonstrated disordered collagen formation similar to keloid tissue.
Conclusion: Our results suggest that under-expression of dermatopontin may play an important role in both fibroid and keloid formation.Decreased dermatopontin may alter both integrin and TGF 3 signaling. Dermatopontin may thereby provide a promoting stimulus for leiomyoma growth. P-393 The correlation of leptin and hCG (Human Chorionic Gonadotrophin) levels in the serum between women with hyperemesis gravidarum and normal control. Jiae Lee, Kyung-Hee Lee, Min-Sik Kim, Bum-Chae Choi, Keun-Jae Yoo, Jeong-Wook Kim. Lab of Reproductive Medicine, Creation & Love Women’s Hosp, Gwangju, Republic of Korea; Ctr for Recurrent Miscarriage and Infertility, Creation & Love Women’s Hosp, Gwangju, Republic of Korea; Dept. of Ob/Gyn, Samsung Cheil Hosp, Sungkyunkwan Univ, Sch of Medicine, Seoul, Republic of Korea.
P ⬍0.05 significance by Mann-Whitney U test. Conclusions: Poor sperm morphology and high SDI are associated with high proportions of apoptotic ⫹ necrotic sperm in ejaculated semen. Thus, the role of morphological assessment cannot be ruled out as a predictor of apoptosis in spermatozoa.
P-392 Promoters of leiomyoma: Dermatopontin expression in leiomyoma suggests an association between fibroids and keloids. William H. Catherino, Matthew H. Stenmark, Phyllis C. Leppert, John C. M. Tsibris, Lynnette K.
FERTILITY & STERILITY威
Objective: Hyperemesis gravidarum is a distinct feature occurring in 0.2-1% of all pregnant women and defined as severe form of nausea and vomiting that lead to fluid and electrolyte imbalance, ketonuria, and weight loss during early pregnancy. Hyperemesis gravidarum may be associated with the development or worsening of an eating disorder during pregnancy. Leptin is the 16kDa protein product of the obese gene, which limits food intake and increases energy expenditure and is produced by the human placenta during pregnancy. High leptin level may decrease food intake and metabolic efficiency. The purpose of this study is to evaluate the correlation of leptin and hCG level in the serum of patients with hyperemesis gravidarum compared with a normal control group. Design: Analysis of serum leptin level by sandwich ELISA and serum hCG level by RIA. Materials and Methods: The serum were collected from 16 women with hyperemesis gravidarum and 10 women with normal pregnancy. They were at the gestation stages of 6-9 weeks. The serum leptin and hCG were
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