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PubMed1991

Utility and predictive value of a rapid measurement of urinary pregnanediol glucuronide by enzyme immunoassay in an infertility practice.

STUDY OBJECTIVE: To assess the clinical utility of measuring urinary pregnanediol glucuronide in random samples in an infertility practice. DESIGN: Samples of urine were collected from patients approximately 3 weeks from their last menstrual period to ascertain if ovulation had occurred. Each sample was tested for specific gravity before analyzing for pregnanediol glucuronide. Simultaneous venipuncture was performed to compare results from the urinary assay to quantitative measures of serum progesterone (P). SETTING: All patients were randomly sampled. PATIENTS: Three hundred ninety women undergoing pituitary down regulation with leuprolide acetate were chosen for study because they routinely initiate medication after documentation of ovulation. OUTCOME MEASURES: The performance of the urinary pregnanediol glucuronide was evaluated as to its sensitivity, specificity, predictive value, and test efficiency compared with a serum measurement of 2.5 ng/mL and 10.0 ng/mL. RESULTS: The performance characteristics of the pregnanediol glucuronide assay were directly related to the hydration status of the patient at the time of sample collection. Regardless of urine specific gravity, if pregnanediol glucuronide was qualitatively detected (greater than 3 micrograms/mL), serum P was greater than 2.5 ng/mL. However, in cases in which pregnanediol glucuronide was undetected (less than 3 micrograms/mL), results were only accurate when the specific gravity was greater than or equal to 1.020. When comparing urinary pregnanediol glucuronide values to serum greater than 10 ng/mL, both specificity and predictive value of a positive test decreased because of increased numbers of false-positive results. CONCLUSIONS: The enzyme immunoassay measurement for pregnanediol glucuronide may replace the use of serum P in documenting the ovulatory status of many patients. However, all specimens must be checked for specific gravity and if less than 1.020, a serum P should be used to ensure accuracy.

Female↗
PubMed1997

LH profile and advancement of ovulation after transcervical infusion of seminal plasma at different stages of oestrus in gilts.

The influence of a transcervical infusion of seminal plasma on preovulatory LH profiles and the advancement of ovulation after seminal plasma infusion for different times during oestrus were investigated using the single uterine horn infusion technique (Mariensee model), in combination with transcutaneous sonographic monitoring of the ovaries. Preparative surgery in 23 German Landrace gilts comprised the detachment of the left uterine horn from the corpus, leaving the caudal end open to the peritoneal cavity but sealing the corpus wound. In six gilts fitted with a permanent jugular vein catheter the patent horns were administered a transcervical infusion of seminal plasma (n = 5 cycles) or PBS (n = 4 cycles) immediately after the detection of oestrus by a teaser boar. In addition, 17 non-catheterized gilts received infusions of seminal plasma either 0 h (n = 3 gilts), 16 h (n = 7 gilts) or 24 h (n = 7 gilts) after the detection of oestrus. Seminal plasma infusion at the onset of oestrus provoked ovulation in the ipsilateral ovary of the treated horn 8.5 +/- 0.9 h earlier than in the contralateral (control) ovary. Seminal plasma did not influence the LH profile compared with PBS (P > 0.05), but shortened the interval between the LH peak and ipsilateral ovulation to 23.4 +/- 4.0 h compared with 31.8 +/- 3.4 h in the contralateral ovulation (P < or = 0.01). Infusion 16 h after the onset of oestrus reduced the effect to 4.6 +/- 3.8 h with a wide range of 0-8 h (P < 0.01). The effect was more pronounced in gilts with long intervals between the onset of oestrus and contralateral ovulation compared with earlier ovulation on the control ovary. Seminal plasma infusion less than 16 h before contralateral ovulation and 24 h after the detection of oestrus had no effect. It is concluded that transcervical infusion of seminal plasma early in oestrus synchronizes the variable intervals between the onset of oestrus and ovulation in sows by a locally active mechanism.

Animals↗
PubMed2003

Luteal activity at the onset of a timed insemination protocol affects reproductive outcome in early postpartum dairy cows.

This study was designed to compare two timed insemination protocols, in which progesterone, GnRH and PGF2alpha were combined, with the Ovsynch protocol in presynchronized, early postpartum dairy cows. Reproductive performance was also evaluated according to whether cows showed high or low plasma progesterone concentration, at the onset of treatment. One hundred and six early postpartum dairy cows were presynchronized with two cloprostenol treatments given 14 days apart, and then assigned to one of the three treatment groups. Treatments for the synchronization of estrus in all three groups started 7 days after the second cloprostenol injection, which was considered Day 0 of the actual treatment regime. Cows in the control group (Ovsynch, n=30) were treated with GnRH on Day 0, PGF2alpha on Day 7, and were given a second dose of GnRH 32 h later. Cows in group PRID (n=45) were fitted with a progesterone releasing intravaginal device (PRID) for 9 days, and were given GnRH at the time of PRID insertion and PGF2alpha on Day 7. In group PRID/GnRH (n=31), cows received the same treatment as in the PRID group, but were given an additional GnRH injection 36 h after PRID removal. Cows were inseminated 16-20 h after the administration of the second GnRH dose in the Ovsynch group, and 56 h after PRID removal in the PRID and PRID/GnRH groups. Ovulation rate was determined on Day 11 postinsemination by detecting the presence of a corpus luteum in the ovaries. Lactation number, milk production, body condition at the onset of treatment and treatment regime were included as potential factors influencing ovulation and pregnancy after synchronization. Logistic regression analysis for cows with high and low progesterone concentration on treatment Day 0 revealed that none of the factors included in the models, except the interaction between progesterone and treatment regime, influenced the risk of ovulation and pregnancy significantly. In cows with high progesterone concentration at treatment onset, Ovsynch treatment resulted in a significantly improved pregnancy rate over values obtained following PRID or PRID/GnRH treatment. In cows with low progesterone concentration, PRID or PRID/GnRH treatment led to markedly increased ovulation and pregnancy rates with respect to Ovsynch treatment. These findings suggest the importance of establishing ovarian status in early postpartum dairy cows before starting a timed AI protocol, in terms of luteal activity assessed by blood progesterone.

Administration, Intravaginal↗
PubMed2004

Post-ovulatory ageing and egg quality: a proteomic analysis of rainbow trout coelomic fluid.

BACKGROUND: In fish, oocyte post-ovulatory ageing is associated with egg quality decrease. During this period, eggs are held in the body cavity where they bath in a semi-viscous liquid known as coelomic fluid (CF). CF components are suspected to play a role in maintaining oocyte fertility and developmental competence (egg quality). However, CF proteic composition remains poorly studied. Thus rainbow trout CF proteome was studied during the egg quality decrease associated with oocyte post-ovulatory ageing. METHODS: High resolution two-dimensional gel electrophoresis was used to analyze the proteome of rainbow trout (Oncorhynchus mykiss) CF in relationship with the egg quality decrease associated with oocyte post-ovulatory ageing. A first experiment was performed using CF pools originating from 17 females sampled at ovulation as well as 7, 14 and 21 days later. These observations were verified using a second set of CF pools originating from 22 females sampled 5 and 16 days following ovulation. RESULTS: Approximately 200 protein spots of 10-105 kDa molecular mass and 3-10 pI were detected in CF samples. Several protein spots, while undetected at the time of ovulation, exhibited a progressive and strong accumulation in CF during post-ovulatory ageing. After silver-staining and Matrix-Assisted Laser Desorption Time Of Flight (MALDI-TOF) mass spectrometer analysis, some of these protein spots were identified as lipovitellin II fragments. CONCLUSIONS: These observations suggest that egg protein fragments accumulate in the CF during the post-ovulatory period and could therefore be used to detect egg quality defects associated with oocyte post-ovulatory ageing.

Aging↗
PubMed1991

[Periovulatory time courses of serum LH in the Japanese monkey (Macaca fuscata)].

Serum LH, E2-17 beta and progesterone concentration were measured in 16 cycles of 15 female Japanese monkeys. Three of the 16 cycles were ascertained to be anovulatory. Ten of the 13 ovulatory cycles showed LH peaks varying from 25 to 280 ng/ml. However, in remaining 3 cycles, LH peak could not be determined, probably because of a lag of blood-sampling schedule. E2-17 beta peaks were detected 0-30 hrs before LH peak in 8 cycles, but 13 or 20 hrs after LH peak in 2 cycles. Time-intervals from LH peak to ovulation ranged 0-47 hrs 30 min. No correlation was detected between concentrations of LH and progesterone in the luteal phase.

Animals↗
PubMed1999

Secretion of zona pellucida glycoprotein mZP2 by growing oocytes from mZP3(+/+) and mZP3(-/-) mice.

The mouse egg extracellular coat, or zona pellucida (ZP), is composed of three glycoproteins, called mZP1-3, which are synthesized and secreted concomitantly by growing oocytes. Disruption of the mZP3 gene by targeted mutagenesis yields mice that are homozygous nulls (mZP3(-/-)). Growing oocytes from mZP3(-/-) mice do not synthesize mZP3 mRNA or protein and, as a result, do not assemble a ZP. Here, we examined secretion of mZP2 by growing oocytes and eggs from mZP3(-/-) mice, as well as incorporation of mZP2 into the ZP of oocytes from mZP3(+/+) mice. Laser scanning confocal microscopy (LSCM) of antibody-labeled samples showed that, indeed, mZP2 was synthesized and secreted by oocytes isolated from mZP3(-/-) mice and cultured in vitro. Nascent mZP2 was found in the culture medium, associated with the surface of the plasma membrane of growing oocytes, and in the oocyte cytoplasm. By contrast, mZP2 was barely detectable at any of these sites when ovulated eggs from mZP3(-/-) mice were examined. Examination of oocytes from wild-type (mZP3(+/+)) mice showed that, while a portion of nascent mZP2 was assembled into the ZP (approximately 40%), here too a significant fraction was secreted into the culture medium (approximately 60%). Similar results also were obtained when intact pre-antral follicles were isolated from mZP3(+/+) mice and cultured in vitro. Several of these observations are consistent with previous results obtained with oocytes from heterozygous null mice (mZP3(+/-)). Furthermore, the results suggest that ZP assembly from nascent glycoproteins may be a stochastic process that requires the presence of both mZP2 and mZP3 and occurs completely outside the growing oocyte.

Animals↗
PubMed1994

Characterization of mouse-hamster-human cross-reacting antioocyte monoclonal antibodies produced by intrasplenic immunization of mice with 12 zona-free mouse oocytes.

Several intrasplenic immunizations with batches of approximately 15 or approximately 30 zona-free, unfertilized mouse oocytes resulted in 200-300 hybrids, respectively, among which about 20 positive clones were selected from each fusion between splenic plasma cells and SP2/0 myeloma cells. When nonimmunized splenic plasma cells were used, only one antibody, showing weak immunoreaction, was obtained from approximately 370 hybrids collected from 2 fusions. From one immunization with a total of 12 zona-free, unfertilized mouse oocytes, 15 positive clones were selected for further study. Eleven of these 15 antibodies reacted with antigens only in unfertilized oocytes but not in fertilized, pronuclear stage oocytes. Three antibodies, which recognized antigens in paraffin-embedded oocyte sections, did not label growing ovarian oocytes, indicating that the antibodies were specific to ovulated, unfertilized oocytes. These antibodies did not detect any antigen epitopes in the panel of tissues examined. The molecular weight of one antigen, corresponding to a IgM antibody that is present both in ooplasma and zona pellucida, was approximately 116 kDa. Cross-reactivity to blots of unfertilized zona-free hamster oocytes was demonstrated by 6 antibodies and to unfertilized human oocytes by 7 antibodies. Three antibodies cross-reacted with both hamster and human oocytes. The study indicates that the intrasplenic immunization is an appropriate means of raising antibodies against unfertilized, zona-free mouse oocytes and that the method applied offers an easy way to select antibodies against human oocytes for functional studies.

Animals↗
PubMed1987

Evaluation of ultrasonography for monitoring follicular growth in rhesus monkeys.

The purpose of this study was to determine if the ovaries and uterus of rhesus monkeys could be visualized by ultrasonography and to detect changes associated with follicular growth and ovulation. Animals were examined during 15 menstrual cycles, for an average of nine consecutive days. Ultrasonic recordings were correlated with hormonal parameters (estradiol 17beta, E(2); luteinizing hormone, LH; and progesterone, P) and laparoscopic findings. The uterus and both ovaries were observed in more than 90% of the examinations. A dominant follicle (DF) was identified during all ovulatory cycles, on average 1 d preceding the E(2) peak. The maximal diameter of the DF ranged from 3 to 7 mm. Laparoscopic examinations to determine the site of the DF confirmed ultrasonic findings in 10 of 14 cycles (P < 0.1). There was no significant difference in the size of the dominant and contralateral ovaries; however, more follicles with a diameter of 2 to 7 mm were found on the dominant ovary (P < 0.05). Two animals stimulated with exogenous gonadotropins showed a linear increase in ovarian size for 6 d prior to oocyte recovery (P < 0.05), reflecting an increase in the number of developing follicles. Ultrasonography can be used to identify the DF during spontaneous cycles in rhesus monkeys and to monitor the response of monkeys to exogenous gonadotropins.

Journal Article↗
PubMed1989

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C.

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 x 10(6) progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20 degrees C or from semen stored for 24 h at 5 degrees C were the same (11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 +/- 2.9, 19.6 +/- 2.6 and 20.5 +/- 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 +/- 2, semen stored for 24 h at 20 degrees C was 57 +/- 11 and semen stored for 24 h at 5 degrees C was 80 +/- 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5 degrees C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20 degrees C than that stored at 5 degrees C.

Journal Article↗
PubMed2005

Specific modulation of nongenomic androgen signaling in the ovary.

Maturation, or meiotic progression, of amphibian oocytes is one of the few physiologically relevant steroid-mediated processes that occurs in the complete absence of transcription from beginning to end. As such, frog oocyte maturation has served as a useful model of nongenomic steroid signaling for many years. Earlier work in Xenopus laevis demonstrated that, although several steroids promoted oocyte maturation in vitro, androgens were the most abundant and potent steroids detected in the serum and ovaries of ovulating frogs. Thus, androgens were likely the primary physiologic regulators of Xenopus oocyte maturation, mediating their actions at least in part via classical androgen receptors expressed in oocytes. The importance of androgens for Xenopus oocyte maturation and ovulation has now been confirmed, as inhibition of androgen production in vivo by blocking CYP17 activity reduced hCG-triggered oocyte maturation and delayed ovulation in female frogs. Taking advantage of the absolute transcription-independence of this androgen-mediated response, selective androgen receptor modulators (SARMs) have been characterized that specifically promote genomic versus nongenomic androgen responses. These include androstenediol and estren, which preferentially promote nongenomic signals, as well as R1881 and 19-nortestosterone, which preferentially promote genomic signaling. Interestingly, the SARMs androstenediol and R1881 signal similarly in mouse oocytes, demonstrating the conserved nature of androgen-mediated maturation in vertebrates. These results suggest that SARMs may serve as useful tools for specifically regulating nongenomic androgen signaling both in vitro and in vivo.

Androgens↗
PubMed1989

Quantitative assessment of the stage of neuronal maturation in the developing human fetal gut--a new dimension in the pathogenesis of developmental anomalies of the myenteric plexus.

There are two main theories of the normal development of the enteric nervous system: the classical theory suggests that the enteric neuroblasts migrate along the alimentary tract in a single cranio-caudal direction. However, the second theory postulates a dual gradient of neuronal development. The present study aims to support the second theory and contribute to the understanding of the pathogenesis of Hirschsprung's disease and its allied gut motility disorders. Using a histomorphometric method, quantitative estimation of the myenteric intraganglionic neuronal development was made in tissues from various selected levels of the gut in 32 normal human fetuses of 11 to 24 weeks ovulation age. A parallel study with special chromatin staining was carried out from these materials to investigate different neurons in the myenteric plexus. Development of intraganglionic neurons was shown to be more advanced in the esophagus, less so in the rectosigmoid and least so in the ileocecal region: the mean values of neuronal and nuclear volume were found in the esophagus greater than in rectosigmoid and ileocecal region as ovulation age progressed. Intraganglionic mitotic figures were detected at all the selected levels of the gut. A decrease of the mitosis index as age progressed was observed. We showed that myenteric neurons followed a dual gradient of development proceeding from both ends to the middle of the gut in mid-trimester human fetuses. Our findings are compatible with the suggestion that any alterations in the fetal gut microenvironment may affect seriously the normal development of a multipotential precursor cell population resulting in various congenital anomalies of the myenteric plexus.

Cell Differentiation↗
PubMed1989

Heat shock protein hsp70 cognate gene expression in vegetative and reproductive organs of Lycopersicon esculentum.

We have detected hsc70 gene expression (heat shock protein hsp70 cognate) during vegetative growth and reproductive development in tomato (Lycopersicon esculentum). Using RNA from a tomato hsc70 cDNA as a probe in in situ hybridizations, we have determined expression patterns of hsc70 in nonstressed tomato roots, stems, leaves, flowers, and developing fruits. We have localized high levels of hsc70 transcript to the vascular system of the ovary, dividing cells of the lateral root tips, and the inner integument of developing seeds. We also see expression in the transmitting tissue, in immature anthers, and in embryos. We cannot detect expression in mature pollen, xylem, or ovules. These data indicate that the expression of at least some tomato hsp70 family members is developmentally regulated.

Journal Article↗
PubMed1999

Insemination at the second of two induced oestrous periods in anoestrous dairy cows increases conception rates to first service.

AIM: To determine whether mating previously anoestrous cows on their second, rather than first, oestrus post pm-turn could increase conception rates to that service. METHODS: Cows diagnosed as anovulatory anoestrus were allocated to two groups, starting treatment on Day -17 (OES2) or on Day -7 (OESI) where the date of the planned start of mating = Day 0. All cows were treated with intravaginal progesterone (CIDR device) for 6 days and an injection of 1 mg oestradiol benzoate 24 h after removal of the device. Cows in the OES2 group were also injected with prostaglandin F2alpha on Day 0. RESULTS: During the first 7 days of mating, 61.8% (207/335) and 88.2% (328/372) of cows in the OES2 and OESl groups, respectively, were inseminated on detection of oestrus (p <0.001). Oestrus without ovulation occurred in 1.5% of cows in the OES2 group and 6.2% of cows in the OESl group during this period (p <0.01). Of those cows inseminated in the first week of mating, conception rates were 54.0% in the OES2 group and 45.5% in the OESl group (p = 0.05). There was no significant difference in median interval from start of mating to conception between the two groups (24 and 22 days for OES2 and OESl, respectively, p >0.05). CONCLUSION: Mating previously anoestrous cows on their second, rather than first oestrus post partum increased conception rates to that service. However, median intervals from start of mating to conception were not altered.

Journal Article↗
PubMed2006

Expression of cyclooxygenase-1 and -2 in the porcine endometrium during the oestrous cycle and early pregnancy.

Cyclooxygenase (COX) is the rate-limiting enzyme that catalyses the initial step in prostaglandins (PGs) production. In the present studies, endometrial COX-1 and COX-2 expression throughout the oestrous cycle and early pregnancy was analysed in pigs using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. There were no changes in messenger RNA (mRNA) and protein expression for COX-1 in cyclic pigs. In pregnant animals, mRNA levels of this enzyme increased on days 22-25 (p < 0.001). However, no upregulation of COX-1 protein was detected. Quantification of COX-2 mRNA expression during the oestrous cycle revealed significant increases on days 10-12 and 14 (p < 0.001 and p < 0.01 vs days 2-4, respectively). Protein levels were also increased on day 14 when compared with days 2-12 and 18-20 after oestrus. In pregnant animals, the patterns of both COX-2 mRNA and protein expression were similar. Messenger RNA levels were higher on days 16 and 22-25 (p < 0.01 vs day 10). Moreover, the protein content tended to increase on days 16 and 22-25. COX-1 and COX-2 were localized in the luminal and glandular epithelium as well as in the uterine stroma. In contrast to COX-1, a positive immunostaining reaction for COX-2 was detected only on days 12-16 after ovulation and on days 14-16 of pregnancy. In conclusion, these results indicate specific patterns of COX-1 and COX-2 expression in the porcine endometrium throughout the oestrous cycle and early pregnancy. COX-2 rather than COX-1 seems to be the primary enzyme responsible for modulated PGs production at the time of luteolysis in cyclic and during implantation in pregnant animals.

Animals↗
PubMed1988

Developmental expression of PDGF, TGF-alpha, and TGF-beta genes in preimplantation mouse embryos.

Control of growth and differentiation during mammalian embryogenesis may be regulated by growth factors from embryonic or maternal sources. With the use of single-cell messenger RNA phenotyping, the simultaneous expression of growth factor transcripts in single or small numbers of preimplantation mouse embryos was examined. Transcripts for platelet-derived growth factor A chain (PDGF-A), transforming growth factor (TGF)-alpha, and TGF-beta 1, but not for four other growth factors, were found in whole blastocysts. TGF-alpha, TGF-beta 1, and PDGF antigens were detected in blastocysts by immunocytochemistry. Both PDGF-A and TGF-alpha were detected as maternal transcripts in the unfertilized ovulated oocyte, and again in blastocysts. TGF-beta 1 transcripts appeared only after fertilization. The expression of a subset of growth factors in mouse blastocysts suggests a role for these factors in the growth and differentiation of early mammalian embryos.

Animals↗
PubMed1992

Delayed implantation. A case report.

In cycles in which conception takes place, serum human chorionic gonadotropin (hCG) becomes detectable between 8 and 12 days after ovulation. A delayed appearance of hCG has been reported in a limited number of cases, most of them ending in spontaneous abortion. We encountered a case of ectopic pregnancy characterized by a delayed appearance of hCG and accompanied by a complete, albeit temporary, halt in the steroidogenic activity of the corpus luteum. Although the patient was at risk of developing an ectopic pregnancy, the findings made an early diagnosis extremely difficult.

Adult↗
PubMed1991

[Ultrasound monitoring of follicle growth in IVF].

Vaginal sonography is a well-established method to monitor follicular growth and to detect the optimum time for induction of ovulation with hCG. In this study we found significantly less and smaller follicles in the left ovary compared to the right. However, during the procedure of oocyte retrieval we found an equal number of follicles and oocytes in both ovaries. We assume therefore that the used sonographic method is less valid for the left ovary compared to the right. The reason could be that on the left side the ovary is often hidden behind the sigma which makes visualization by ultrasound more difficult. Vaginal sonography should therefore be performed on a gynecological chair where it is easier to bring the probe closer to the left ovary. On the basis of follicular fluid volume measurements at the puncture the sizes of the follicles on day hCG were calculated and resulted in a range of 14.7-20.7 mm for the average of all follicles.

Chorionic Gonadotropin↗
PubMed1985

[Value of the measurement of SP1 protein in the early diagnosis of pregnancy induced with Pregnyl].

Successive dilutions of Pregnyl have been submitted to a radioimmunoassay of SP1. We have observed that Pregnyl cross-reacts with SP1. Chromatography of Pregnyl demonstrates that the contaminating substance is different from native SP1. As its concentration is low, it is however not detected in the serum of patients after the administration of exogenous Pregnyl. SP1 measurement can be helpful in detecting early pregnancies after hCG induction of ovulation.

Adult↗